CN105658236A - Antibodies - Google Patents

Abstract

The present invention relates to an anti-CSF-1R antibody and binding fragments thereof, DNA encoding the same, host cells comprising said DNA and methods of expressing the antibody or binding fragment in a host cell. The present invention also extends to pharmaceutical compositions comprising the antibody or a binding fragment thereof and use of the antibody, binding fragment and compositions comprising the same in treatment.

Description

Antibody

technical field

The present invention relates to anti-CSF-1R antibodies and binding fragments thereof, DNA encoding anti-CSF-1R antibodies and binding fragments thereof, host cells comprising said DNA and methods for expressing said antibodies or binding fragments in host cells. The invention also extends to pharmaceutical compositions comprising said antibodies or binding fragments thereof, and the use of said antibodies, binding fragments and compositions comprising said antibodies, binding fragments in therapy.

Background technique

Colony-stimulating factor 1 (CSF-1 ), also known as macrophage colony-stimulating factor (M-CSF), is a cytokine produced by various cells, including macrophages, endothelial cells, and fibroblasts. CSF-1 comprises two "monomeric" polypeptides that form the biologically active dimeric CSF-1 protein. CSF-1 exists in at least three mature forms due to alternative RNA splicing (see Cerretti et al., 1988, Molecular Immunology, 25:761). These three forms of CSF-1 are translated from different rnRNA precursors that encode polypeptide monomers of 256 to 554 amino acids with an amino-terminal 32 amino acid signal sequence and a carboxy-terminal approximately 23 amino acid putative transmembrane region. Subsequently, the precursor peptide is processed by amino-terminal and carboxy-terminal proteolytic cleavage to release mature CSF-1. Residues 1-149 of all three mature forms of CSF-1 are identical and are believed to contain sequences essential for the biological activity of CSF-1. In vivo, CSF-1 monomers are glycosylated and dimerized via disulfide bonds. CSF-1 belongs to a group of biological agonists that promote the production of blood cells. Specifically, it acts as a growth and differentiation factor for myeloid progenitor cells of the mononuclear phagocyte lineage. In addition, CSF-1 stimulates macrophage survival, proliferation and function through specific receptors on responsive cells.

CSF-1 receptor (CSF-1R) is also known as the c-fms gene product or CD115. CSF-IR is a 165 kDa type 1 TM glycoprotein that belongs to the type III receptor tyrosine kinase family. It has also been shown that, in addition to CSF-1, the structurally similar but sequence-unrelated molecule IL-34 is also a ligand for CSF-1R (Lin et al., 2008, Science 320:807-811). Expression of CSF-IR is restricted to cells of the monocyte-macrophage lineage (circulating and tissue-resident population) as well as osteoclasts. In addition, it is expressed in many cells of the female reproductive system, including oocytes, decidual cells, and trophoblast cells.

Binding of the ligand CSF-1 to the CSF-1 receptor leads to phosphorylation of the receptor on one or more tyrosine residues through the action of the tyrosine kinase domain. This phosphorylation can be detected because antibodies are available that bind only the phosphorylated receptor (eg Phospho-M-CSF-Receptor (Tyr546) antibody #3083 from Cell Signaling Technology).

Expression of CSF-1 and CSF-1R is associated with tumor progression and poor diagnosis in many cancer types. Tumor-associated macrophages (TAMs) can be a major component of the tumor stroma and high levels of CSF-1 and CSF-1R correlate with high TAM infiltration and poor prognosis in many tumor types.

Antibodies against CSF-IR are known in the art. Sherr, C.J. et al., 1989, Blood 73:1786-1793 describe an anti-CSF-1R antibody that inhibits CSF-1 activity (Sherr, C.J. et al., 1989, Blood 73:1786-1793). WO2009/026303 discloses anti-CSF-1R antibodies that bind human CSF-1R and in vivo mouse tumor models using anti-mouse CSF-1R antibodies. WO2011/123381 discloses anti-CSF-1R antibodies that internalize CSF-1R and have ADCC activity. WO2011/123381 also discloses an in vivo mouse tumor model using an anti-mouse CSF-1R antibody. WO2011/140249 discloses anti-CSF-1R antibodies that block the binding of CSF-1 to CSF-1R, which are believed to be useful in the treatment of cancer. WO2009/112245 discloses anti-CSF-1 RIgG1 antibodies that inhibit CSF-1 binding to CSF-1R, which are believed to be useful in the treatment of cancer, inflammatory bowel disease and rheumatoid arthritis. WO2011/131407 discloses anti-CSF-1R antibodies that inhibit CSF-1 binding to CSF-1R, which are believed to be useful in the treatment of bone loss and cancer. WO2011/107553 discloses anti-CSF-1R antibodies that inhibit CSF-1 binding to CSF-1R, which are believed to be useful in the treatment of bone loss and cancer. WO2011/070024 discloses anti-CSF-1R antibodies that bind to the human CSF-1R fragment delD4.

There is a need in the art to provide novel anti-CSF-1R antibodies suitable for use in therapeutic applications. Although the therapeutic use of anti-CSF-1R antibodies in the treatment of certain cancers has been previously described, there remains a need to provide novel therapeutic uses of such antibodies.

The term "fibrotic disease" refers to an abnormal wound healing response in which excess fibrous connective tissue forms in an organ or tissue. Deposition and accumulation of excess extracellular matrix components, such as collagen and fibronectin, leads to hardening and scarring of tissues, which can eventually lead to organ failure.

Examples of fibrotic diseases include pulmonary fibrosis such as idiopathic pulmonary fibrosis and cystic fibrosis, renal fibrosis, liver fibrosis, liver cirrhosis, primary sclerosing cholangitis, primary biliary cirrhosis, cardiac Intimal myocardial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, Crohn's disease, keloids, myocardial infarction, systemic sclerosis , scleroderma, and joint fibrosis.

Trauma leads to an immediate coagulation and coagulation response with the development of a provisional extracellular matrix (ECM). Platelet aggregation and activation help promote an inflammatory response characterized by vasodilation and increased vascular permeability, thereby recruiting a variety of immune cells including neutrophils, macrophages, eosinophils, and lymphocytes. Neutrophils and macrophages debride the wound, thereby reducing the risk of infection, and together with activated lymphocytes, secrete various growth factors and cytokines that further amplify the inflammatory response. Molecules such as TGFβ, PDGF, IL-13 activate macrophages and lead to the recruitment, proliferation and activation of fibroblasts at the wound site. Activated fibroblasts or myofibroblasts are characterized by the expression of [alpha]-smooth muscle actin and the secretion of collagen and other ECM components. Activated fibroblasts contract the collagen lattice, pulling the edges of the wound toward the center. Epidermal and endothelial cells proliferate and migrate on a provisional matrix to regenerate damaged tissue, thereby completing wound repair.

Persistent tissue injury or damage, or dysregulation of repair pathways leads to inappropriate wound responses. Excessive deposition and hypercrosslinking of collagen and ECM occurs, resulting in excessive formation and hardening of scar tissue that replaces normal tissue architecture.

The etiology of fibrotic disease can depend on the organ or tissue involved and is unknown in some diseases, such as idiopathic pulmonary fibrosis (IPF). Liver fibrosis and eventually cirrhosis result from persistent chronic liver injury through exposure to various factors including environmental and dietary factors or infectious agents. Long-term hepatitis B or C infection can lead to liver fibrosis. Continued excessive consumption of alcohol or a high-fat/sugar diet can also lead to hardening of the liver. Similarly, diabetes can damage and scar the kidneys, resulting in loss of function.

IPF is one of seven interstitial lung diseases of unknown etiology. Environmental factors such as radiation exposure or particles may play a role. Subjects who smoked were also at higher risk of developing the disease. After diagnosis, patients have an extremely short lifespan, with an average survival rate of 2-5 years.

Treatment of fibrotic diseases often includes anti-inflammatory and immunosuppressive agents, but these are of less benefit to the patient. The lack of efficacy of these treatments has led to the general reconsideration of IPF and fibrotic diseases as abnormal responses to wound healing rather than inflammatory conditions. Pirfenidone, a small molecule drug approved for the treatment of IPF in Japan in 2008 and in Europe in 2011, may act through multiple mechanisms of action. To date, no targeted and antibody therapies have been approved for fibrosis indications.

Accordingly, there currently exists an unmet medical need for improved treatments for fibrotic diseases. For example, with IPF, the 3-year survival rate is 50% and the 5-year survival rate is only 20%, and transplantation is required in approximately 20% of cases.

Summary of the invention

In one aspect, an anti-CSF-1R antibody or binding fragment thereof comprising a heavy chain is provided, wherein the variable domain of the heavy chain comprises a CDR having the sequence shown in SEQ ID NO: 4 for CDR-H1, having a sequence for CDR-H2 has at least one of the CDR of the sequence shown in SEQ ID NO: 5 and the CDR with the sequence shown in SEQ ID NO: 6 for CDR-H3, for example, wherein CDR-H1 is SEQ ID NO: 4, CDR-H2 is SEQ ID NO: 5 and CDR-H3 is SEQ ID NO: 6.

In one aspect, an antibody or binding fragment according to the present disclosure comprises a light chain, wherein the variable domain of the light chain comprises a CDR having the sequence shown in SEQ ID NO: 1 for CDR-L1, having a sequence shown in SEQ ID NO: 1 for CDR-L2. At least one of the CDR of the sequence shown in :2 and the CDR having the sequence shown in SEQ ID NO: 3 for CDR-L3, for example, wherein CDR-L1 is SEQ ID NO: 1, CDR-L2 is SEQ ID NO: 2 and CDR-L3 is SEQ ID NO:3.

The antibodies of the disclosure have high affinity for CSF-1R, are able to block ligand binding to CSF-1R, do not activate CSF-1R, and do not cause internalization of CSF-1R.

The disclosure also extends to polynucleotides (such as DNA) encoding antibodies or fragments as described herein.

Also provided is a host cell comprising the polynucleotide.

Also provided herein are methods of expressing antibodies or binding fragments thereof.

The present disclosure also relates to pharmaceutical compositions comprising said antibodies or binding fragments thereof.

In one embodiment, there is provided a method of treatment comprising administering a therapeutically effective amount of an antibody, fragment or composition as described herein.

The present disclosure also extends to the use of the antibodies, binding fragments or compositions of the present disclosure in therapy, in particular in the treatment of cancer and/or fibrotic diseases.

Detailed description of the invention

In one embodiment, antibodies provided by the invention are capable of blocking ligand binding to CSF-1R. As used herein, blocking refers to physical blocking, such as blocking a receptor, but also includes situations where an antibody or fragment binds an epitope, which causes, for example, a conformational change, meaning that the natural ligand of the receptor no longer binds (referred to herein as allosteric blocking or allosteric inhibition). In one embodiment, antibodies of the disclosure bind to all isotypes of CSF-1R, e.g., with alterations in the ECD domain (such as V23G, A245S, H247P, V279M, and two, three of said alterations or a combination of four) of those.

Suitable assays for determining the ability of an antibody to block CSF-IR are described in the Examples herein. Both CSF-1 and IL-34 are ligands for CSF-1R and the antibodies of the invention preferably inhibit the activity of both CSF-1 and IL-34 in functional cell screens. The antibodies of the invention also preferably do not lead to CSF-1R activation and/or CSF-1R internalization. Antibodies of the invention also preferably selectively deplete atypical populations of monocytes in vivo.

Atypical monocytes generally refer to monocytes with low CD14 expression and high CD16 expression. This monocyte population is thought to be the precursor of tumor-associated macrophages.

Antibody molecules of the invention suitably have high binding affinity. As described in the Examples herein, isolated native or recombinant CSF-1R or an appropriate fusion protein/polypeptide may be used using any suitable method known in the art, including techniques such as surface plasmon resonance (e.g., BIAcore) to measure affinity. In one example, affinity is measured using recombinant human CSF-IR ectodomain as described in the Examples herein. In one example, the recombinant human CSF-1R ectodomain used is a monomer. Suitably, the antibody molecule of the invention has a binding affinity for isolated human CSF-IR of about 1 nM or less. In one embodiment, an antibody molecule of the invention has a binding affinity of about 500 pM or less. In one embodiment, an antibody molecule of the invention has a binding affinity of about 250 pM or less. In one embodiment, an antibody molecule of the invention has a binding affinity of about 200 pM or less. In one embodiment, the invention provides an anti-CSF-1R antibody with a binding affinity of about 100 pM or less. In one embodiment, the present invention provides a humanized anti-CSF-1R antibody with a binding affinity of about 100 pM or less (preferably about 10 pM or less, more preferably about 5 pM or less). In another embodiment, the present invention provides a humanized anti-CSF-1R antibody with a binding affinity of about 100 pM or less (preferably about 10 pM or less, more preferably about 5 pM or less).

The lower the numerical value for affinity, the greater the affinity of the antibody or fragment for the antigen.

As used herein, human CSF-1R refers to the human protein designated CSF-1R or a biologically active fragment thereof, for example, as shown in SEQ ID NO: 39 or registered in UniProt under accession number P07333. Of course, the expressed mature protein does not contain a signal sequence because the signal sequence is cleaved after translation.

The present inventors have provided novel anti-CSF-1R antibodies, including humanized antibodies. Antibodies were produced from immunization of rats with rat fibroblasts transfected with a vector expressing the CSF-1R ectodomain. Primary screening of supernatants for human CSF-1R binding of antibodies identified approximately 1000 wells containing antibodies with anti-CSF-1R activity. A secondary screen for antibodies capable of preventing binding of human CSF-1 to human CSF-IR identified 88 positive wells. Tertiary screening for antibodies capable of preventing CSF-1-dependent survival of primary human monocytes identified 18 positive wells. Cloning of the variable regions of these 18 positive wells resulted in the successful cloning of 14 antibodies and subsequent expression provided 9 chimeric anti-CSF-1R antibodies expressed at sufficient levels and capable of inhibiting CSF-1 binding. These 9 antibodies were sequenced and found to all have unique sequences and were used for further studies.

These nine anti-CSF-1R chimeric antibodies were evaluated for their ligand-blocking activity and ability to inhibit CSF-1- and IL-34-mediated monocyte survival. Four of the antibodies were prioritized for further studies as they demonstrated complete inhibition of CSF-1 binding and high levels of inhibition of monocyte survival. The activity of these 4 anti-CSF-1R chimeric antibodies was tested in a number of in vitro assays to assess affinity, inhibition of CSF-1 binding, interaction with rhesus monkey (rhesus monkey), cynomolgus monkey (cynomolgus monkey) and canine CSF-1R Cross-reactivity, CSF-1R internalization, and CSF-1R activation. These four anti-CSF-1R antibodies were also humanized and the affinities of the humanized grafts were measured. Humanization of two of the anti-CSF-1R antibodies resulted in a fully humanized antibody with an affinity ( _KD ) equal to that of the parental chimeric antibody and a Tm indicative of the appropriate thermal stability of the antibody (in the absence of a rat donor). body residues). In contrast, the other two humanized anti-CSF-1R antibodies had reduced affinity for CSF-1R and smaller Tm relative to the chimeric antibody. For these reasons, only affinity-retaining fully humanized grafts of two of the antibodies were expressed on a larger scale for further analysis.

Further analyzes of fully humanized grafts of these two antibodies were performed and MCP-1 inhibition assays were performed in which inhibition of CSF-1R signaling by antibodies that block CSF-1 binding resulted in reduced levels of MCP-1 secretion. This assay surprisingly showed that the fully humanized graft exhibited reduced activity compared to the chimeric antibody. A series of experiments was performed on a preferred antibody (termed Ab969) to reveal why fully humanized grafts exhibited this reduced activity. A number of intermediate humanized grafts of Ab969 were generated and tested in the MCP-1 inhibition assay. Grafts containing the variable light chain donor residue Y71 were found to generally show comparable activity to chimeric Ab969 in MCP-1 inhibition assays. It has been hypothesized that the difference in activity of the various antibody grafts in the MCP-1 inhibition assay is due to a change in antibody on-rate (reduced Ka) compared to the chimeric _Ab969 .

A comparison of the thermostability analysis of Ab969 with other anti-CSF-1R antibodies, such as anti-CSF-1R antibody Ab970, shows that Ab969 may be more stable.

Further biophysical analysis of the humanized grafts of Ab969 showed that some grafts precipitated upon antibody concentration. Substitution of the lysine residue at position 38 in the light chain (eg glutamine) has been shown to result in improved physical stability.

Therefore, one antibody graft of Ab969, Ab969.g2 (also referred to as Ab969), was selected for further in vitro characterization studies. In addition to favorable high CSF-1R binding affinity, high thermal stability and high physical stability, the antibody exhibited good inhibition of IL-34-dependent monocyte activation and was also found to be able to bind SNP variants of CSF-1R. Ab969.g2 was also used in a pharmacodynamic marker assay in cynomolgus monkeys, where Ab969.g2 was shown to bind CSF-1R, block CSF-1 binding, and selectively deplete atypical cynomolgus monocytes in vivo population, which are precursor cells of tumor-associated macrophages.

Thus, in one embodiment, the invention provides an antibody comprising a heavy chain and/or a light chain comprising at least one CDR derived from anti-CSF-1R antibody 969.2.

Ab969.2 is a full-length humanized IgG4 molecule; the light chain contains the human kappa chain constant region (Km3 allotype) and the heavy chain contains the human gamma-4 heavy chain constant with the hinge stabilizing mutation S241P (Angal et al., 1993). Area. A potential DG isomerization motif exists at the junction of CDR-L2 and the framework in the light chain variable region. The sequences of Ab969.2 full antibody heavy and light chains are shown in SEQ ID NO:27 and 19.

The residues in the variable domains of antibodies are conventionally numbered according to the system devised by Kabat et al., 1987 . This system is shown in Kabat et al., 1987, Sequences of Proteins of Immunological Interest, USD Department of Health and Human Services, NIH, USA (hereinafter "Kabat et al. (supra)"). This numbering system is used in this specification unless otherwise indicated.

The Kabat residue nomenclature does not always correspond directly to the linear numbering of amino acid residues. The actual linear amino acid sequence may contain fewer or additional amino acids than in strict Kabat numbering, which correspond to shortenings or insertions of structural components (whether framework or complementarity determining regions (CDRs)) of the base variable domain structure. The correct Kabat numbering of residues for a given antibody can be determined by an alignment between the homologous residues in the antibody sequence and the "standard" Kabat numbering sequence.

According to the Kabat numbering system, the CDRs of the heavy chain variable domain are located at residues 31-35 (CDR-H1), residues 50-65 (CDR-H2) and residues 95-102 (CDR-H3). However, according to Chothia (Chothia, C. and Lesk, A.M., J. Mol. Biol., 196, 901-917 (1987)), the loop equivalent to CDR-H1 extends from residue 26 to residue 32. Thus, unless otherwise indicated, "CDR-H1" as used herein means residues 26 to 35, as described by combining the Kabat numbering system and the Chothia topological ring definition.

According to the Kabat numbering system, the CDRs of the light chain variable domain are located at residues 24-34 (CDR-L1), residues 50-56 (CDR-L2) and residues 89-97 (CDR-L3).

Antibodies for use in the present disclosure can be obtained using any suitable method known in the art. CSF-1R polypeptides/proteins (including fusion proteins), cells expressing the polypeptides (recombinantly or naturally) can be used to generate antibodies that specifically recognize CSF-1R. A polypeptide may be a "mature" polypeptide or a biologically active fragment or derivative thereof. The human protein is registered in UniProt under accession number P07333.

Polypeptides for immunizing hosts can be produced from genetically engineered host cells, including expression systems, by methods well known in the art, or they can be recovered from natural biological sources. In this application, the term "polypeptide" includes peptides, polypeptides and proteins. These terms are used interchangeably unless otherwise indicated. In some cases, a CSF-1R polypeptide may be part of a larger protein (such as a fusion protein, eg, fused to an affinity tag, etc.).

Where it is necessary to immunize the animal, antibodies raised against the CSF-1R polypeptide can be obtained by administering the polypeptide to animals (preferably non-human animals) using well known and routine protocols, see e.g. Handbook of Experimental Immunology, D.M. Weir (ed.) , Vol4, Blackwell Scientific Publishers, Oxford, England, 1986. Many warm-blooded animals such as rabbits, mice, rats, sheep, cows, camels or pigs can be immunized. However, mice, rabbits, pigs and rats are generally most suitable.

Monoclonal antibodies can be prepared by any method known in the art, such as hybridoma technology (Kohler & Milstein, 1975, Nature, 256:495-497), triorioma technology, human B-cell hybridoma technology ( Kozbor et al., 1983, Immunology Today, 4:72) and EBV-hybridoma technology (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc.).

Antibodies can also use the monolymphocyte antibody method, for example by Babcook, J. et al., 1996, Proc. Said method is produced by cloning and expressing immunoglobulin variable region cDNA produced from single lymphocytes selected for specific antibody production.

Screening of antibodies can be performed using assays that measure binding to human CSF-IR and/or assays that measure the ability to block ligand binding to the receptor. Examples of suitable assays are described in the Examples herein.

As used herein, "specificity" refers to an antibody that only recognizes the antigen it is specifically directed against or has a significantly higher binding affinity for the antigen it is specifically directed against, as compared to binding to an antigen it is non-specifically directed against, e.g. At least 5, 6, 7, 8, 9, 10 fold higher binding affinities.

The amino acid sequences and polynucleotide sequences of certain antibodies of the disclosure are provided in FIGS. 1 and 2 .

In one aspect of the invention, the antibody is an anti-CSF-1R antibody or binding fragment thereof comprising a heavy chain, wherein the variable domain of the heavy chain comprises a CDR having the sequence shown in SEQ ID NO: 4 for CDR-H1, having At least one of the CDR with the sequence shown in SEQ ID NO: 5 for CDR-H2 and the CDR with the sequence shown in SEQ ID NO: 6 for CDR-H3. Preferably, the variable domain of the heavy chain comprises the sequence shown in SEQ ID NO: 4 for CDR-H1, the sequence shown in SEQ ID NO: 5 for CDR-H2 and the sequence shown in SEQ ID NO: 6 for CDR-H3 sequence.

In a second aspect of the invention, the antibody is an anti-CSF-1R antibody or binding fragment thereof comprising a light chain, wherein the variable domain of the light chain comprises a CDR having the sequence shown in SEQ ID NO: 1 for CDR-L1, At least one of the CDR having the sequence shown in SEQ ID NO: 2 for CDR-L2 and the CDR having the sequence shown in SEQ ID NO: 3 for CDR-L3. Preferably, the variable domain of the light chain comprises the sequence shown in SEQ ID NO: 1 for CDR-H1, the sequence shown in SEQ ID NO: 2 for CDR-H2 and the sequence shown in SEQ ID NO. 3 for CDR-H3 sequence.

In one embodiment, the antibody of the invention is an anti-CSF-1R antibody or a binding fragment thereof comprising a heavy chain as defined above and additionally comprising a light chain, wherein the variable domain of the light chain comprises At least one of the CDR having the sequence shown in :1, the CDR having the sequence shown in SEQ ID NO: 2 for CDR-L2, and the CDR having the sequence shown in SEQ ID NO: 3 for CDR-L3. The variable domain of the light chain preferably comprises the sequence shown in SEQ ID NO: 1 for CDR-L1, the sequence shown in SEQ ID NO: 2 for CDR-L2 and the sequence shown in SEQ ID NO: 3 for CDR-L3.

In one embodiment, at least one amino acid in one or more CDRs selected from the group independently consisting of is replaced by a conservative substitution:

Any of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3;

Combined CDR-H1 and H2, CDR-H1 and H3, CDR-H1 and L1, CDR-H1 and L2, CDR-H1 and L3, CDR-H2 and H3, CDR-H2 and L1, CDR-H2 and L2, CDR - any of H2 and L3, CDR-H3 and L1, CDR-H3 and L2, CDR-H3 and L3, CDR-L1 and L2, CDR-L1 and L3, CDR-L2 and L3;

CDR-H1, H2 and H3, CDR-H1, H2 and L1, CDR-H1, H2 and L2, CDR-H1, H2 and L3, CDR-H2, H3 and L1, CDR-H2, H3 and L2, CDR- H2, H3 and L3, CDR-H3, L1 and L2, CDR-H3, L1 and L3, CDR-L1, L2, L3;

Combined CDR-H1, H2, H3 and L1, CDR-H1, H2, H3 and L2, CDR-H1, H2, H3 and L3, CDR-H2, H3, L1 and L2, CDR-H2, H3, L2 and L3 , CDR-H3, L1, L2 and L3, CDR-L1, L2, L3 and H1, CDR-L1, L2, L3 and H2, CDR-L1, L2, L3 and H3, CDR-L2, L3, H1 and H2 any of

CDR-H1, H2, H3, L1 and L2, CDR-H1, H2, H3, L1 and L3, CDR-H1, H2, H3, L2 and L3, CDR-L1, L2, L3, H1 and H2, CDR- L1, L2, L3, H1 and H3, CDR-L1, L2, L3, H2 and H3; and

Combine CDR-H1, H2, H3, L1, L2 and L3.

In one embodiment, the domain of the heavy chain disclosed herein comprises a sequence with 1, 2, 3 or 4 conservative amino acid substitutions, eg, wherein the substitutions are in frame.

In one embodiment, the framework of the heavy chain variable region comprises 1, 2, 3, or 4 amino acids that have been inserted, deleted, substituted, or combinations thereof. In one embodiment, the substituted amino acid is the corresponding amino acid from the donor antibody.

In one embodiment, the light chain variable region disclosed herein comprises a sequence with 1, 2, 3 or 4 conservative amino acid substitutions, eg, wherein the substitutions are in frame.

In one embodiment, the framework of the light chain variable region comprises 1, 2, 3 or 4 amino acids that have been inserted, deleted, substituted or combinations thereof. In one embodiment, the substituted amino acid is the corresponding amino acid from the donor antibody.

In one aspect of the present invention, an anti-CSF-1R antibody or a binding fragment thereof is provided, wherein the variable domain of the heavy chain comprises three kinds of CDRs and the sequence of CDR-H1 has at least 60 %, 70%, 80%, 90% or 95% identity or similarity, the sequence of CDR-H2 has at least 60%, 70%, 80%, 90% or 95% identity with the sequence shown in SEQ ID NO:5 Identity or similarity and the sequence of CDR-H-3 is at least 60%, 70%, 80%, 90% or 95% identical or similar to the sequence shown in SEQ ID NO:6. Preferably, the anti-CSF-1R antibody or binding fragment thereof additionally comprises a light chain, wherein the variable domain of the light chain comprises three CDRs and the sequence of CDR-L1 has at least 60%, 70%, 80%, 90% or 95% identity or similarity, the sequence of CDR-L2 has at least 60%, 70%, 80%, 90% or 95% identity or similarity to the sequence shown in SEQ ID NO: 2 And the sequence of CDR-L3 has at least 60% identity or similarity with the sequence shown in SEQ ID NO:3.

In one embodiment, provided variable regions are at least 60%, 70%, 80%, 90% or 95% identical or similar to the variable region sequences disclosed herein. In another embodiment, there is provided an antibody or fragment of the invention that competes for binding to the CSF-1R receptor (preferably the extracellular domain of the CSF-1R receptor), more specifically binding to a protein having SEQ ID NO: 35, 36, 37, 38 and/or 39 or a CSF-1R receptor of the sequence of UniProt database entry P07333, specifically binding to the ectodomain of the CSF-1R receptor having SEQ ID NO: 36 or an ectodomain disclosed in UniProt database entry P07333 Anti-CSF-1R antibody of 498 amino acids (amino acids 20 to 517 of P07333).

In one embodiment, there is provided a cross-blocking sequence comprising SEQ ID NO: 1 for CDR-L1, SEQ ID NO: 2 for CDR-L2, SEQ ID NO: 3 for CDR-L3, and SEQ ID NO: 3 for CDR-H1 An anti-CSF-1R antibody binding, for example, with an affinity of 100 pM or less, of antibodies against the six CDRs shown in SEQ ID NO: 4, for CDR-H2 with SEQ ID NO: 5, and for CDR-H3 with SEQ ID NO: 6, particularly , wherein said cross-blocking is allosteric.

In another embodiment, there is provided an anti-CSF-1R-antibody or binding fragment thereof that inhibits the binding of CSF-1 and/or IL-34 to the extracellular domain of the CSF-1R receptor or to CSF-1 and and/or binding of IL-34 to the extracellular domain of the CSF-1R receptor overlaps.

In one embodiment, there is provided a cross-blocker comprising SEQ ID NO: 1 for CDR-L1, SEQ ID NO: 2 for CDR-L2, SEQ ID NO: 3 for CDR-L3, and SEQ ID NO: 3 for CDR-H1. SEQ ID NO: 4, an anti-CSF-1R antibody that binds, for example, with an affinity of 100 pM or less, for antibodies against the six CDRs shown in SEQ ID NO: 5 for CDR-H2 and SEQ ID NO: 6 for CDR-H3, in particular, wherein said antibody cross-blocks binding by binding to the same epitope as the antibody it blocks.

In one embodiment, an antibody or binding fragment thereof is provided wherein the C-terminal residue of the antibody sequence (eg, the C-terminal residue of the heavy chain sequence, eg, the terminal lysine) is cleaved. In one embodiment, this amino acid is cleaved from the sequences disclosed herein. In general, cleavage results from post-translational modifications of expressed antibodies or binding fragments.

In another embodiment, there is provided the anti-CSF-1R antibody of any of the above or following embodiments, wherein the C-terminal lysine of the heavy chain sequence shown in SEQ ID NO: 27 or SEQ ID NO: 29 is deleted or deleted . A missing or deleted C-terminal lysine (e.g., position 453 of SEQ ID NO: 27 or position 472 of SEQ ID NO: 30) can be expressed, for example, by expressing an anti-CSF-1R antibody in an expression system without encoding a terminal lysine to realise. Alternatively, deletion of C-terminal residues (such as lysine) can be achieved as a post-translational modification.

In one embodiment, an antibody or binding fragment of the invention is humanized.

As used herein, the term "humanized antibody" refers to an antibody or antibody molecule in which the heavy and/or light chains contain elements from a donor antibody (e.g., a murine monoclonal antibody) grafted into a recipient antibody (e.g., One or more CDRs (including one or more modified CDRs if desired) in the heavy and/or light chain variable region framework of a human antibody) (see eg US5,585,089; WO91/09967). For a review see Vaughan et al., Nature Biotechnology, 16 , 535-539, 1998. In one embodiment, only one or more specificity determining residues from any of the CDRs described herein above, rather than the entire CDR, are transferred to the human antibody framework (see e.g. Kashmiri et al., 2005, Methods, 36:25- 34). In one embodiment, only the specificity determining residues from one or more of the CDRs described herein above are transferred to the human antibody framework. In another embodiment, only the specificity determining residues from each of the CDRs described herein above are transferred to the human antibody framework. When grafting CDRs or specificity determining residues, any suitable recipient variable region framework sequence may be used, including mouse, primate and human frameworks, taking into account the species/type of donor antibody from which the CDRs are derived Area.

Suitably, a humanized antibody of the invention has a variable domain comprising a human acceptor framework region and one or more CDRs as specifically provided herein. Accordingly, in one embodiment there is provided a humanized antibody that binds human CSF-1R, wherein the variable domain comprises a human acceptor framework region and a non-human donor CDR.

Examples of human frameworks that can be used in the present invention are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Kabat et al., supra). For example, KOL and NEWM can be used for heavy chain, REI can be used for light chain and EU, LAY and POM can be used for heavy chain and light chain. Alternatively, human germline sequences can be used; these are available at http://vbase.mrc-cpe.cam.ac.uk/ .

In the humanized antibodies of the invention, the acceptor heavy and light chains are not necessarily derived from the same antibody and may, if desired, comprise composite chains with framework regions derived from different chains.

In one embodiment, the human framework comprises 1, 2, 3, or 4 amino acid substitutions, additions, or deletions, eg, 1, 2, 3, or 4 conservative substitutions of donor residues.

In one embodiment, the sequences used as human frameworks have 80%, 85%, 90%, 95% or more similarity or identity to the sequences disclosed herein.

One such suitable framework region for the heavy chain of a humanized antibody of the invention is derived from the human subgroup VH2 sequence 3-12-70 plus the JH3J-region (SEQ ID NO: 33).

Therefore, in one example, a method comprising the sequence shown in SEQ ID NO: 4 for CDR-H1, the sequence shown in SEQ ID NO: 5 for CDR-H2 and the sequence shown in SEQ ID NO: 6 for CDR-H3 is provided. A humanized antibody of the sequence shown, wherein the heavy chain framework region is derived from the human subgroup VH3 sequence 1-33-07 plus JH4.

In one example, the heavy chain variable domain of the antibody comprises the sequence set forth in SEQ ID NO:23.

Suitable framework regions for the light chains of the humanized antibodies of the invention are derived from the human germline subgroup VK12-1-(1)O12 plus the JK4J-region (SEQ ID NO: 31).

Therefore, in one example, a method comprising the sequence shown in SEQ ID NO: 1 for CDR-L1, the sequence shown in SEQ ID NO: 2 for CDR-L2 and the sequence shown in SEQ ID NO: 3 for CDR-L3 is provided. Humanized antibody of the sequence shown, wherein the light chain framework region is derived from human subgroup VK12-1-(1)O12 plus JK4J-region.

In one example, the light chain variable domain of the antibody comprises the sequence set forth in SEQ ID NO:15.

In the humanized antibody of the present invention, the framework region does not need to have the exact same sequence as that of the acceptor antibody. For example, uncommon residues can be changed to residues that are more common to that class or type of acceptor chain. Alternatively, selected residues in the acceptor framework regions can be altered such that they correspond to residues found at the same positions in the donor antibody (see Reichmann et al., 1998, Nature, 332:323-324). Such alterations should be kept to the minimum necessary to restore the affinity of the donor antibody. A protocol for selecting residues in the framework regions of an acceptor that may need to be altered is shown in WO 91/09967.

Thus, in one example, a humanized antibody is provided wherein at least the residue at position 78 (Kabat numbering) of the heavy chain variable domain is a donor residue. In one embodiment, residue 78 of the heavy chain variable domain is replaced with alanine.

Donor residues as used herein refer to residues from a non-human antibody (eg, a murine antibody) that donated the CDRs.

In one embodiment, a humanized antibody is provided wherein the heavy chain variable domain does not comprise any donor residues.

Thus, in one example, a humanized antibody is provided wherein at least one of the residues at positions 38, 71 and 87 (Kabat numbering) of the light chain variable domain is a donor residue. In one embodiment, one of the residues selected from the residues at positions 38, 71 and 87 (Kabat numbering) of the light chain variable domain is a donor residue. In one embodiment, two of the residues selected from the residues at positions 38, 71 and 87 (Kabat numbering) of the light chain variable domain (eg 38 and 71; or 38 and 87; or 71 and 87 ) is the donor residue. In one embodiment, the three residues at positions 38, 71 and 87 (Kabat numbering) of the light chain variable domain are donor residues.

In one embodiment, residue 38 of the light chain variable domain is replaced with lysine. In an alternative embodiment, residue 38 of the light chain variable domain is replaced with glutamine.

In one embodiment, residue 71 of the light chain variable domain is replaced with tyrosine.

In one embodiment, residue 87 of the light chain variable domain is replaced with phenylalanine.

In one embodiment, a humanized antibody is provided wherein only residue 71 in the light chain variable region is a donor residue, preferably tyrosine.

In specific embodiments, the present invention provides an anti-CSF-1R antibody or binding fragment thereof, which has a heavy chain comprising a heavy chain variable domain sequence represented by SEQ ID NO: 23 and comprising a heavy chain variable domain sequence represented by SEQ ID NO: 15 The light chain of the light chain variable domain sequence.

In other aspects of the present disclosure, there is provided an anti-CSF-1R antibody or binding fragment thereof that binds CSF-1R (preferably the extracellular domain of CSF-1R, most preferably the extracellular domain of human CSF-1R), wherein the antibody Or the binding fragment comprises the light chain variable domain of SEQIDNO: 15 and the heavy chain variable domain of SEQIDNO: 23, preferably, wherein the antibody or binding fragment thereof is a monoclonal antibody, IgG1-IgG2-, IgG4-type antibody, Fab , modified Fab, Fab', modified Fab', F(ab') ₂ , Fv, single domain antibody (eg, VH or VL or VHH), scFv, bivalent, trivalent or tetravalent antibody, Bispecific - scFv, diabody, triabody or tetrabody.

In one embodiment, the disclosure provides a sequence that is 80% similar or identical to a sequence disclosed herein (e.g., 85%, 90%, 91%, 92%, 93%, 94%, over part or all of a related sequence) 95%, 96%, 97%, 98%, or 99% similar or identical) to the antibody sequence. In one embodiment, the related sequence is SEQ ID NO:15. In one embodiment, the related sequence is SEQ ID NO:23.

As used herein, "identity" indicates that at any particular position in the aligned sequences, the amino acid residues between the sequences are identical. As used herein, "similarity" indicates that at any particular position in the aligned sequences, the amino acid residues between the sequences are of a similar type. For example, leucine can be replaced with isoleucine or valine. Other amino acids that can generally be substituted for each other include, but are not limited to:

- phenylalanine, tyrosine and tryptophan (amino acids with aromatic side chains);

- lysine, arginine and histidine (amino acids with basic side chains);

- aspartic acid and glutamic acid (amino acids with acidic side chains);

- asparagine and glutamine (amino acids with amide side chains); and

- cysteine and methionine (amino acids with sulfur-containing side chains). Degrees of identity and similarity can be easily calculated (Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987, Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991, BLAST ^TM software Available from NCBI (Altschul, SF et al., 1990, J. Mol. Biol. 215:403-410; Gish, W. & States, DJ 1993, Nature Genet. 3:266-272. Madden, TL et al., 1996, Meth. Enzymol. 266:131-141; Altschul, SF et al., 1997, Nucleic Acids Res. 25:3389-3402; Zhang, J. & Madden, TL 1997, Genome Res. 7:649-656).

Antibody molecules of the invention may include complete antibody molecules with full length heavy and light chains or binding fragments thereof and may be, but are not limited to, Fab, modified Fab, Fab', modified Fab', F(ab') _2. Fv, single domain antibody (such as VH or VL or VHH), scFv, bivalent, trivalent or tetravalent antibody, bispecific-scFv, diabody, triabody, tetrabody and epitopes of any of the above antibodies Binding fragments (see eg Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, DrugDesignReviews-Online 2(3), 209-217). Methods for creating and manufacturing these antibody fragments are well known in the art (see eg Verma et al., 1998, Journal of Immunological Methods, 216:165-181). Other antibody fragments useful in the present invention include Fab and Fab' fragments described in International Patent Applications WO05/003169, WO05/003170 and WO05/003171. Multivalent antibodies may comprise multiple specificities such as bispecifics or may be monospecific (see eg WO92/22853, WO05/113605, WO2009/040562 and WO2010/035012).

A binding fragment of an antibody as used herein refers to a fragment that is capable of binding an antigen with an affinity that characterizes the fragment as being specific for the antigen.

In one embodiment, an antibody of the disclosure is provided as a CSF-1R binding antibody fusion protein comprising an immunoglobulin portion (e.g., a Fab or Fab' fragment) and one or two single domains directly or indirectly linked thereto Antibodies (dAbs), eg as described in WO2009/040562, WO2010/035012, WO2011/030107, WO2011/061492 and WO2011/086091, all incorporated herein by reference.

In one embodiment, the fusion protein comprises two domain antibodies, for example as a variable heavy (VH) and variable light (VL) pair, optionally linked by a disulfide bond.

In one embodiment, the Fab or Fab' element of the fusion protein has the same or similar specificity as a single domain antibody. In one embodiment, the Fab or Fab' has a different specificity than the single domain antibody, in other words the fusion protein is multivalent. In one embodiment, the multivalent fusion protein according to the invention has an albumin binding site, for example, wherein the VH/VL pair provides an albumin binding site.

In one embodiment, the Fab or Fab' member of the fusion protein has the same or similar specificity as that of the single domain antibody. In one embodiment, the Fab or Fab' pairs or the single domain antibodies have different specificities, in other words the fusion protein is multivalent. In one embodiment, the multivalent fusion proteins of the disclosure have an albumin binding site, eg, where the VH/VL pair provides an albumin binding site.

The constant region domains, if any, of the antibody molecules of the invention can be selected based on the proposed function of the antibody molecule and, in particular, effector functions that may be required. For example, the constant region domains may be human IgA, IgD, IgE, IgG or IgM domains. In particular, human IgG constant region domains, particularly of the IgG1 and IgG3 isotypes, may be used when the antibody molecule is intended for therapeutic use and antibody effector functions are desired. Alternatively, IgG2 and IgG4 isotypes can be used when the antibody molecule is intended for therapeutic purposes and antibody effector functions are not required.

In specific embodiments, the antibodies of the invention are IgG2 or IgG4 antibodies.

It will be appreciated that sequence variants of this constant region domain may also be used. For example, an IgG4 molecule in which the serine at position 241 has been changed to proline as described in Angal et al., 1993, Molecular Immunology, 1993, 30: 105-108 can be used. Thus, in embodiments wherein the antibody is an IgG4 antibody, the antibody may include the mutation S241P.

Those of skill in the art will also appreciate that antibodies can undergo various post-translational modifications. The type and extent of these modifications often depend on the host cell line and culture conditions used to express the antibody. Such modifications may include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization, and asparagine deamidation. A common modification is the deletion of a carboxy-terminal basic residue such as lysine or arginine due to the action of carboxypeptidases (as described in Harris, RJ. Journal of Chromatography 705:129-134, 1995). Thus, the C-terminal lysine of the antibody heavy chain may be absent.

In one embodiment, the antibody heavy chain comprises a CH1 domain and the antibody light chain comprises a CL domain (kappa or lambda).

In one embodiment, the antibody heavy chain comprises a CH1 domain, a CH2 domain and a CH3 domain, and the antibody light chain comprises a CL domain (kappa or lambda).

The antibody provided by the present invention has a heavy chain comprising the sequence shown in SEQ ID NO:27 and a light chain comprising the sequence shown in SEQ ID NO:19. Also provided is an anti-CSF-1R antibody or binding fragment thereof, wherein the heavy chain and light chain are at least 80% identical to the heavy chain comprising the sequence shown in SEQ ID NO: 27 and the light chain comprising the sequence shown in SEQ ID NO. 19 (preferably 85%, 90%, 95% or 98%) the same or similar. In one embodiment, the light chain has or consists of the sequence set forth in SEQ ID NO: 19 and the heavy chain has or consists of the sequence set forth in SEQ ID NO: 27. In another embodiment, the light chain has or consists of the sequence of SEQ ID NO: 19 and the heavy chain has or consists of the sequence of SEQ ID NO: 27, wherein the amino acid lysine at position 453 of SEQ ID NO: 27 is deleted or deleted.

The present disclosure also provides the specificity of human CSF-1R bound by the antibody provided by the invention (specifically, the antibody 969.g2 comprising the heavy chain sequence gH2 (SEQ ID NO: 27) and/or the light chain sequence gL7 (SEQ ID NO: 19)). Sexual regions or epitopes.

The specific region or epitope of the human CSF-1R polypeptide can be identified by any suitable epitope mapping method known in the art in combination with any antibody provided in the present invention. Examples of such methods include screening peptides of varying lengths (e.g., peptides in the range of about 5 to 10 (preferably about 7) amino acids in length) derived from CSF-1R for binding to the antibodies of the invention, wherein specific The smallest fragment that sexually binds the antibody contains the sequence of the epitope recognized by the antibody. CSF-1R peptides can be produced synthetically or by proteolytic digestion of CSF-1R polypeptides. Peptides that bind antibodies can be identified, for example, by mass spectrometry. In another example, NMR spectroscopy or X-ray crystallography can be used to identify epitopes bound by antibodies of the invention. After identification, fragments of an epitope that bind an antibody of the invention can be used as an immunogen, if desired, to obtain additional antibodies that bind the same epitope.

Biomolecules, such as antibodies or fragments, contain acidic and/or basic functional groups, giving the molecule a net positive or negative charge. The amount of "observed" total charge will depend on the absolute amino acid sequence of the entity, the local environment of the charged groups in the 3D structure and the environmental conditions of the molecule. The isoelectric point (pi) is the pH at which a particular molecule or its solvent-accessible surface has no net charge. In one example, CSF-1R antibodies and fragments of the invention can be engineered to have an appropriate isoelectric point. This may result in antibodies and/or fragments having more potent properties, in particular suitable solubility and/or stability characteristics and/or improved purification properties.

Thus, in one aspect, the invention provides a humanized CSF-1R antibody engineered to have an isoelectric point different from that of the originally identified antibody. Antibodies can be engineered, for example, by substituting amino acid residues, such as substituting one or more basic amino acid residues for an acidic amino acid residue. Alternatively, basic amino acid residues may be introduced or acidic amino acid residues may be removed. Alternatively, acidic residues can be introduced to lower the pi if the molecule has an unacceptably high pi value, if desired. Importantly, when manipulating the pi, care must be taken to preserve the desired activity of the antibody or fragment. Thus, in one embodiment, the engineered antibody or fragment has the same or substantially the same activity as the "unmodified" antibody or fragment.

Programs such as **ExPASY http://www.expasy.ch/tools/pi_tool.html and http://www.iut-arles.up.univ-mrs.fr/w3bb/d_abim/compo-p.html can be Used to predict the isoelectric point of an antibody or fragment.

It will be appreciated that the affinity of the antibodies provided by the invention may be altered using any suitable method known in the art. Accordingly, the present invention also relates to variants of the antibody molecules of the invention, which have improved affinity for CSF-1R. Such variants can be obtained by a number of affinity maturation protocols, including mutating CDRs (Yang et al., 1995, J. Mol. Biol., 254:392-403), chain shuffling (Marks et al., 1992, Bio/Technology, 10 :779-783), using mutant strains of Escherichia coli (Low et al., 1996, J.Mol.Biol., 250:359-368), DNA shuffling (Patten et al., 1997, Curr.Opin.Biotechnol., 8 :724-733), phage display (Thompson et al., J.Mol.Biol., 256,77-88, 1996) and sexual PCR (sexualPCR) (Crameri et al., 1998, Nature, 391 :288-291) . These affinity maturation methods are discussed by Vaughan et al. (supra).

Antibodies for use in the invention can be conjugated to one or more effector molecules, if desired. It will be appreciated that an effector molecule may comprise a single effector molecule or two or more such molecules linked to form a single moiety which can be attached to an antibody of the invention. When it is desired to obtain an antibody fragment linked to an effector molecule, this can be prepared by standard chemistry or recombinant DNA procedures, wherein the antibody fragment is linked to the effector molecule either directly or via a coupling agent. Techniques for conjugating such effector molecules to antibodies are well known in the art (see Hellstrom et al., Controlled Drug Delivery, 2nd Ed., Robinson et al., eds., 1987, pp. 623-53; Thorpe et al., 1982, Immunol. Rev., 62:119-58 and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123). Specific chemistries include, for example, those described in WO93/06231, WO92/22583, WO89/00195, WO89/01476 and WO03/031581. Alternatively, where the effector molecule is a protein or polypeptide, linkage can be achieved using recombinant DNA procedures (eg as described in WO86/01533 and EP0392745).

The term "effector molecule" as used herein includes, for example, antineoplastic agents, drugs, toxins, biologically active proteins such as enzymes, other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof such as DNA, RNA Fragments thereof, radionuclides, especially radioiodides, radioisotopes, chelated metals, nanoparticles and reporter groups such as fluorescent compounds or compounds detectable by NMR or ESR spectroscopy.

Examples of effector molecules may include cytotoxins or cytotoxic agents, including any agent that is detrimental to (eg, kills) cells. Examples include combrestatin, dolastatin, epothilone, staurosporine, maytansinoids, spongistatin, rhizoxin, halichondrin, bacitracin, hemiasterlins, Paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, dauna Erythromycin, dihydroxyanthraxedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine , propranolol and puromycin and their analogs or homologues.

Effector molecules also include, but are not limited to, antimetabolites (e.g. methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine), alkylating agents (e.g. nitrogen mustard, thioguanine Tepa, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide (cyclothosphamide), busulfan, dibromomannitol, streptozotocin, mitomyces C and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (such as daunorubicin (formerly daunorubicin) and doxorubicin), antibiotics (such as dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthranimycin (AMC), calicheamicin, or duocarmycins) and antimitotic agents (eg, vincristine and vinblastine).

Other effector molecules may include chelated radionuclides such as ¹¹¹ In and ⁹⁰ Y, Lu ¹⁷⁷ , Bismuth ²¹³ , Californium ²⁵² , Iridium ¹⁹² and Tungsten ¹⁸⁸ /Rhenium ¹⁸⁸ ; or drugs such as but not limited to alkylphosphorylcholines, topoisotropic Constructase I inhibitors, taxanes and suramin.

Other effector molecules include proteins, peptides and enzymes. Enzymes of interest include, but are not limited to, proteolytic enzymes, hydrolases, lyases, isomerases, transferases. Proteins, polypeptides and peptides of interest include but are not limited to immunoglobulins, toxins such as abrin, ricin A, pseudomonas exotoxin or diphtheria toxin, proteins such as insulin, tumor necrosis factor, alpha-interferon , beta-interferon, nerve growth factor, platelet-derived growth factor or tissue plasminogen activator, thrombotic or anti-angiogenic agents such as angiostatin or endostatin, or biological response modifiers such as lymphokines, interleukins -1 (IL-1), interleukin-2 (IL-2), nerve growth factor (NGF) or other growth factors and immunoglobulins.

Other effector molecules may include, for example, detectable substances useful in diagnostics. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radionuclides, positron-emitting metals (for positron emission tomography), and non-radioactive paramagnetic metals ion. See generally US Patent No. 4,741,900 for metal ions that can be conjugated to antibodies for use as diagnostic agents. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin, and biotin; suitable fluorescent Materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin; suitable luminescent materials include luminol; suitable Bioluminescent materials include luciferase, luciferin, and aequorin; and suitable radionuclides ^include125I , ^131I , ^{111In and99Tc} .

In another example, the effector molecule can increase the in vivo half-life of the antibody, and/or reduce the immunogenicity of the antibody and/or enhance delivery of the antibody across the epithelial cell barrier to the immune system. Examples of suitable effector molecules of this type include polymers, albumin, albumin binding proteins or albumin binding compounds such as those described in WO05/117984.

In one embodiment, the half-life provided by effector molecules independent of CSF-IR is advantageous.

In general, where the effector molecule is a polymer, it may be a synthetic or naturally occurring polymer, such as an optionally substituted linear or branched polyalkylene, polyalkenylene or polyoxyalkylene base polymers or branched or unbranched polysaccharides, such as homopolysaccharides or heteropolysaccharides.

Particular optional substituents that may be present on the synthetic polymers described above include one or more hydroxy, methyl or methoxy groups.

Specific examples of synthetic polymers include optionally substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol) or derivatives thereof, especially optionally substituted poly(ethylene glycol) ) such as methoxypoly(ethylene glycol) or derivatives thereof.

Specific naturally occurring polymers include lactose, amylose, dextran, glycogen or derivatives thereof.

In one embodiment, the polymer is albumin or a fragment thereof, such as human serum albumin or a fragment thereof.

"Derivative" as used herein is intended to include reactive derivatives, for example thiol-selective reactive groups such as maleimide and the like. The reactive group can be attached to the polymer directly or via a linker segment. It will be appreciated that residues of such groups will in some cases form part of the product as linking groups between the antibody fragment and the polymer.

The size of the polymer may vary as desired, but will generally be in the range of average molecular weight 500 Da to 50000 Da, eg 5000 to 40000 Da such as 20000 to 40000 Da. The size of the polymer can be chosen based on the intended use of the product, eg the ability to localize to certain tissues such as tumors or prolong the circulating half-life (for a review see Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531-545). Thus, for example, the use of small molecular weight polymers (eg having a molecular weight of about 5000 Da) may be advantageous when the product is intended to leave the circulation and penetrate tissue (eg for the treatment of tumors). For applications where the product remains in circulation, it may be advantageous to use higher molecular weight polymers, for example having a molecular weight in the range of 20000 Da to 40000 Da.

Suitable polymers include polyalkylene polymers such as poly(ethylene glycol), or especially methoxypoly(ethylene glycol) or derivatives thereof, and especially have a molecular weight in the range of about 15,000 Da to about 40,000 Da .

In one example, antibodies for use in the invention are attached to poly(ethylene glycol) (PEG) moieties. In a specific example, the antibody is an antibody fragment and the PEG molecule can be attached via any available amino acid side chain or terminal amino acid functional group (e.g., any free amino, imino, sulfhydryl, hydroxyl, or carboxyl) located in the antibody fragment. Such amino acids may occur naturally in antibody fragments or may be engineered into fragments by using recombinant DNA methods (see eg US5,219,996; US5,667,425; WO98/25971, WO2008/038024). In one example, an antibody molecule of the invention is a modified Fab fragment, wherein the modification is the addition of one or more amino acids to the C-terminus of its heavy chain to allow attachment of effector molecules. Suitably, the additional amino acids form a modified hinge region containing one or more cysteine residues to which the effector molecule may be attached. Multiple sites can be used to attach two or more PEG molecules.

Suitably, the PEG molecule is covalently linked via a sulfhydryl group located at at least one cysteine residue in the antibody fragment. Each polymer molecule attached to the modified antibody fragment can be covalently linked to a sulfur atom of a cysteine residue located in that fragment. The covalent linkage will generally be a disulfide bond or especially a sulfur-carbon bond. Where sulfhydryl groups are used as attachment points, appropriately activated effector molecules may be used, eg thiol-selective derivatives such as maleimide and cysteine derivatives. Activated polymers can be used as starting materials in the preparation of polymer-modified antibody fragments as described above. The activated polymer can be any polymer containing sulfhydryl-reactive groups such as alpha-halocarboxylic acids or esters such as iodoacetamide, imides such as maleimide, vinyl sulfone or disulfides. things. Such starting materials are either commercially available (eg from Nektar (formerly Shearwater Polymers Inc.), Huntsville, AL, USA) or can be prepared from commercially available starting materials using conventional chemical methods. Specific PEG molecules include 20K methoxy-PEG-amine (available from Nektar, formerly Shearwater; Rapp Polymere; and SunBio) and M-PEG-SPA (available from Nektar, formerly Shearwater).

In one embodiment the antibody is a modified Fab fragment, Fab' fragment or diFab which is PEGylated, i.e. has PEG (poly(ethylene glycol)) covalently attached thereto, for example according to EP0948544 or EP1090037 The disclosed method (see also "Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical Applications", 1992, J. Milton Harris (ed), Plenum Press, New York, "Poly(ethyleneglycol) Chemistry and Biological Applications", 1997, J. Milton Harris and S. Zalipsky (eds), American Chemical Society , Washington DC and "Bioconjugation Protein Coupling Techniques for the Biomedical Sciences", 1998, M. Aslamand A. Dent, Grove Publishers, New York; Chapman, A. 2002, Advanced Drug Delivery Reviews 2002, 54:531-545). In one example, PEG is attached to cysteines in the hinge region. In one example, the PEG-modified Fab fragment has a maleimide group covalently linked to a single sulfhydryl group in the modified hinge region. A lysine residue can be covalently linked to a maleimide group and a methoxy poly(ethylene glycol) polymer having a molecular weight of about 20,000 Da can be attached to each of the lysine residues. Amino. Thus, the total molecular weight of PEG attached to the Fab fragment can be about 40,000 Da.

Specific PEG molecules include 2-[3-(N-maleimido)propionamide] of N,N'-bis(methoxypoly(ethylene glycol) MW 20,000) modified lysine Acetamide, also known as PEG2MAL40K (available from Nektar, formerly Shearwater).

Alternative sources of PEG linkers include NOF, which supplies GL2-400MA3 (where m is 5 in the structure below) and GL2-400MA (where m is 2) and n is about 450:

That is, each PEG is approximately 20,000 Da.

Thus, in one embodiment PEG is 2,3-bis(methylpolyoxyethylene-oxy)-1-{[3-(6-maleimido-1-oxohexyl)amino]propoxy }Hexane (2-arm branched PEG, -CH ₂ ) ₃ NHCO(CH ₂ ) ₅ -MAL, Mw 40,000, called SUNBRIGHTGL2-400MA3.

Further optional PEG effector molecules of the following types:

In one embodiment, the invention provides an antibody, such as a full-length antibody, that is PEGylated (e.g., with PEG as described herein) through the amino acid at or about amino acid position 226 of the chain (e.g., at amino acid position 226 of the heavy chain). Cysteine amino acid residues at position amino acids (by consecutive numbering)) are attached.

In one embodiment, the present disclosure provides Fab'PEG molecules comprising one or more PEG polymers, for example 1 or 2 polymers such as a 40 kDa polymer.

Fab-PEG molecules according to the present disclosure may be particularly advantageous because they have a half-life independent of the Fc fragment.

In one embodiment, the invention provides scFv conjugated to a polymer such as a PEG molecule, a starch molecule or an albumin molecule.

In one embodiment, the antibody or fragment is conjugated to a starch molecule, for example to increase half-life. Methods of conjugating starch to proteins are described in US 8,017,739, which is incorporated herein by reference.

A reporter molecule, as used herein, is a molecule capable of being detected, eg, a fluorescent dye, radioactive label, or other detectable entity.

The invention also provides isolated DNA sequences encoding the heavy and/or light chains of the antibody molecules of the invention. Suitably, the DNA sequence encodes the heavy or light chain of an antibody molecule of the invention. The DNA sequences of the invention may include, for example, synthetic DNA, cDNA, genomic DNA, or any combination thereof produced by chemical processing.

The DNA sequence encoding the antibody molecule of the present invention can be obtained by methods well known to those skilled in the art. For example, DNA sequences encoding part or all of antibody heavy and light chains can be synthesized as desired from defined DNA sequences or based on corresponding amino acid sequences.

DNAs encoding acceptor framework sequences are widely available to those skilled in the art and can be readily synthesized based on their known amino acid sequences.

Standard techniques of molecular biology can be used to prepare DNA sequences encoding the antibody molecules of the invention. The desired DNA sequence can be synthesized in whole or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be suitably employed.

Examples of suitable DNA sequences are provided in Figure 1 .

The invention also relates to cloning or expression vectors comprising one or more DNA sequences of the invention. Thus, cloning or expression vectors comprising one or more DNA sequences encoding the antibodies of the invention are provided. In one embodiment, the vector comprises the sequence set forth in SEQ ID NO:28 and/or SEQ ID NO:20. Suitably, the cloning or expression vector comprises two DNA sequences (preferably SEQ ID NO: 28 and SEQ ID NO: 20, respectively) encoding the light and heavy chains of the antibody molecule of the invention, respectively, and an appropriate signal sequence. In one example, the vector comprises an intergenic sequence between the heavy and light chains (see WO03/048208).

General methods for constructing vectors, transfection methods and culture methods are well known to those skilled in the art. In this regard, reference is made to "Current Protocols in Molecular Biology", 1999, F.M. Ausubel (ed), Wiley Interscience, New York and the Maniatis Manual, published by Cold Spring Harbor Publishing.

Also provided are host cells comprising one or more cloning or expression vectors comprising one or more DNA sequences encoding the antibodies of the invention. Any suitable host cell/vector system can be used for expression of the DNA sequences encoding the antibody molecules of the invention. Bacterial (eg, E. coli) and other microbial systems can be used, or eukaryotic (eg, mammalian) host cell expression systems can also be used. Suitable mammalian host cells include CHO, myeloma or hybridoma cells.

The invention also provides a method of making an antibody molecule of the invention comprising culturing a host cell comprising a vector of the invention under conditions suitable to result in protein expression from DNA encoding the antibody molecule of the invention, and isolating the antibody molecule.

Antibody molecules may comprise only heavy or light chain polypeptides, in which case only heavy or light chain polypeptide coding sequences need be used to transfect host cells. For production of products comprising heavy and light chains, two vectors (a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide) can be used to transfect a cell line. Alternatively, a single vector can be used that includes sequences encoding both heavy and light chain polypeptides.

Antibodies and fragments of the disclosure are expressed at good levels from host cells. Thus, the properties of the antibodies and/or binding fragments are suitable for expression on a commercial scale.

Accordingly, methods of culturing host cells and expressing antibodies or fragments thereof, isolating antibodies or fragments thereof, and optionally purifying them to provide isolated antibodies or fragments are provided. In one embodiment, the method further comprises the step of conjugating an effector molecule to the isolated antibody or fragment, eg, to a PEG polymer, in particular as described herein.

In one embodiment, there is provided a method of purifying an antibody, particularly an antibody or fragment of the invention, comprising performing anion exchange chromatography in non-binding mode such that impurities are retained on the column and the antibody is eluted.

In one embodiment, purification utilizes affinity capture on a CSF-1R column.

In one embodiment, purification utilizes cibacron blue or the like for purification of albumin fusion or conjugate molecules.

Suitable ion exchange resins for use in this method include Q.FF resin (supplied by GE-Healthcare). The steps can be performed, for example, at a pH of about 8.

The method may further comprise an initial capture step using cation exchange chromatography, eg at a pH of about 4 to 5, such as 4.5. Cation exchange chromatography may for example use a resin such as CaptoS resin or SP Sepharose FF (supplied by GE-Healthcare). Antibodies or fragments can then be eluted from the resin using, for example, an ionic salt solution such as sodium chloride at a concentration of 200 mM.

Thus, a chromatography step may suitably comprise one or more washing steps.

Purification methods may also include one or more filtration steps, such as diafiltration steps.

Thus in one embodiment there is provided a purified anti-CSF-1R antibody or fragment, e.g., a humanized antibody or fragment, in particular an antibody or fragment of the invention, in substantially purified form, in particular free of or Essentially free of endotoxin and/or host cell proteins or DNA.

Purified form as used above means at least 90% pure, such as 91, 92, 93, 94, 95, 96, 97, 98, 99% w/w or higher.

Substantially free of endotoxin generally means an endotoxin content of 1 EU/mg antibody product or less, such as 0.5 or 0.1 EU/mg product.

Substantially free of host cell protein or DNA generally means a host cell protein and/or DNA content of suitably 400 μg/mg antibody product or less, such as 100 μg/mg or less, especially 20 μg/mg.

The present invention also provides an anti-CSF-1R antibody of the disclosure (or a pharmaceutical composition comprising the same) for use as a medicament.

The present invention also provides an anti-CSF-1R antibody of the disclosure (or a pharmaceutical composition comprising the same) for use in the treatment of cancer.

The present invention also provides the use of the anti-CSF-1R antibody of the present disclosure (or the pharmaceutical composition comprising it) in the preparation of a medicament for treating or preventing cancer.

The invention also provides a method of treating a human subject having or at risk of cancer, the method comprising administering to the subject an effective amount of an anti-CSF-1R antibody of the disclosure.

Antibodies of the disclosure can be used to treat cancers selected from the group consisting of breast cancer, prostate cancer, bone cancer, myeloma, colorectal cancer, leukemia, lymphoma, skin cancer (such as melanoma), esophageal cancer, gastric cancer, star cancer Cell carcinoma, endometrial cancer, cervical cancer, bladder cancer, kidney cancer, lung cancer, liver cancer, thyroid cancer, head and neck cancer, pancreatic cancer and ovarian cancer.

In one embodiment, the cancer is a metastatic cancer from any of the primary cancers listed above, especially bone cancer.

Surprisingly, we have been able to demonstrate that anti-CSF-1R antibodies that inhibit CSF-1R activity are active in the treatment of fibrotic diseases. Specifically, we have been able to demonstrate that anti-CSF-1R antibodies are active in an in vivo animal model of pulmonary fibrosis.

The present invention also provides an anti-CSF-1R antibody of the present disclosure (or a pharmaceutical composition comprising the same) for use in the treatment of fibrotic diseases.

The present invention also provides the use of the anti-CSF-1R antibody of the present disclosure (or the pharmaceutical composition comprising it) in the preparation of a medicament for treating or preventing fibrotic diseases.

The present invention also provides a method for treating a human subject having or at risk of a fibrotic disease, the method comprising administering to the subject an effective amount of an anti-CSF-1R antibody of the disclosure.

In this application, the term "fibrotic disease" includes diseases characterized by an abnormal response to wound healing in which excess fibrous connective tissue forms in an organ or tissue. Exemplary fibrotic diseases include, but are not limited to, pulmonary fibrosis such as idiopathic pulmonary fibrosis and cystic fibrosis, renal fibrosis including renal tubular atrophy and interstitial fibrosis, liver fibrosis, liver cirrhosis, primary cirrhosis cholangitis, primary biliary cirrhosis, endomyocardial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, Crowe Graces, keloids, myocardial infarction, scleroderma, systemic sclerosis, and joint fibrosis.

In one embodiment, an antibody or fragment of the disclosure is used to treat or prevent cancer or a fibrotic disease.

Antibodies of the disclosure can be used to treat inflammatory diseases such as inflammatory arthritis, atherosclerosis, multiple sclerosis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, rheumatoid spondylitis, Ankylosing arthritis, arthritis, psoriatic arthritis, rheumatoid arthritis, osteoarthritis, eczema, contact dermatitis, psoriasis, toxic shock syndrome, sepsis, septic shock, endotoxic shock, Asthma, chronic pneumonia, silicosis, pulmonary sarcoidosis, osteoporosis, restenosis, cardiac and renal reperfusion injury, thrombosis, glomerulonephritis, diabetes, graft-versus-host reaction, Allograft rejection, multiple sclerosis, muscle degeneration, muscular dystrophy, Alzheimer's disease, and stroke.

Antibodies and fragments of the disclosure may be used therapeutically or prophylactically.

Antibody molecules of the invention may also be used in diagnostics, eg, in vivo diagnosis and imaging of disease states involving CSF-1R.

Because the antibody of the present invention can be used for the treatment and/or prevention of pathological conditions, the present invention also provides pharmaceuticals comprising the antibody molecule of the present invention in combination with one or more of pharmaceutically acceptable excipients, diluents or carriers. Pharmaceutical or diagnostic composition. Therefore, the use of the antibody of the present invention for the preparation of a medicament is provided. The compositions will generally be presented as part of a sterile pharmaceutical composition which will generally comprise a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention may additionally contain a pharmaceutically acceptable adjuvant.

The invention also provides a method of preparing a pharmaceutical or diagnostic composition comprising adding and mixing an antibody molecule of the invention and one or more of a pharmaceutically acceptable excipient, diluent or carrier.

Antibody molecules may be the sole active ingredient in a pharmaceutical or diagnostic composition. Alternatively, an antibody may be administered in combination (eg, simultaneously, sequentially, or separately) with one or more other therapeutically active ingredients. Accordingly, the antibody molecule in the pharmaceutical or diagnostic composition may be accompanied by other active ingredients including other antibody components, for example, epidermal growth factor receptor family (EGFR, HER-2), vascular endothelial growth factor receptor Antibody (VEGFR), platelet-derived growth factor receptor (PDGFR), or non-antibody components such as imatinib, dasatinib, nioltinib, basutinib (basutinib), gefitinib, erlotinib, temsirolimus, vandetanib, vemurafenib, crizotinib ), vorinostat, romidepsin, bortezomib, sorafenib, sunitinib, pazopanib, regorafex regorafenib, cabozantinib, pirfenidone, steroids or other drug molecules (especially drug molecules whose half-life is independent of CSF-1R binding).

Active ingredient as used herein refers to an ingredient that has a pharmacological effect, such as a therapeutic effect, at the relevant dosage.

The pharmaceutical composition suitably comprises a therapeutically effective amount of an antibody of the invention. The term "therapeutically effective amount" as used herein refers to the amount of a therapeutic agent required to treat, ameliorate or prevent a target disease or condition, or exhibit a detectable therapeutic, pharmacological or prophylactic effect. For any antibody, the therapeutically effective amount can be estimated first in cell culture assays or in animal models, typically rodents, rabbits, dogs, pigs or primates. Animal models can also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes of administration in humans.

The precise therapeutically effective amount for a human subject will depend on the severity of the condition, the general health of the subject, the age, weight and sex of the subject, diet, time and frequency of administration, drug combination, response, Sensitivity and tolerance/response to treatment. Such amounts can be determined by routine experimentation and are within the judgment of a physician. Generally, a therapeutically effective amount will be from 0.01 mg/kg to 500 mg/kg, eg, from 0.1 mg/kg to 200 mg/kg, such as 100 mg/kg.

The pharmaceutical compositions may conveniently be presented in unit dosage form containing a predetermined amount of an active agent of the invention per dosage.

Therapeutic doses of the antibodies of the disclosure showed no apparent or limited in vivo toxicological effects.

Compositions may be administered to a patient alone or may be administered in combination (eg, simultaneously, sequentially or separately) with other agents, drugs or hormones.

In one embodiment, an antibody or binding fragment of the disclosure is used with one or more other cancer treatment options such as chemotherapy, radiation therapy or surgery. If administered with a chemotherapeutic agent, the antibody may be administered before or after or concurrently with the chemotherapeutic agent. Chemotherapy treatments that can be used in combination with the provided antigen binding proteins include but are not limited to alkylating/DNA damaging agents (e.g. carboplatin, cisplatin), antimetabolites (e.g. capecitabine, gemcitabine, 5-fluorouracil) . Mitotic inhibitors (eg paclitaxel, vincristine).

Antibodies to be used in the treatment of various inflammatory diseases can be used alone or in combination with various other anti-inflammatory agents.

Antibodies to be used in the treatment of various fibrotic diseases can be used alone or in combination with various other anti-fibrotic agents. An example of such an agent is Perfedidone.

The administered dose of the antibody molecule of the invention depends on the nature of the condition to be treated, the severity of the condition presented and on whether the antibody molecule is used prophylactically or for the treatment of an existing condition.

The frequency of dosing will depend on the half-life of the antibody molecule and the duration of its effect. If the antibody molecule has a short half-life (eg, 2 to 10 hours), one or more daily doses may be required. Alternatively, if the antibody molecule has a long half-life (e.g., 2 to 15 days) and/or long-lasting pharmacodynamic (PD) properties, it may only require dosing once a day, once a week, or even every 1 or 2 months once.

Half-life as used herein means the duration of a molecule in circulation eg serum/plasma.

Pharmacodynamics as used herein refers to the profile and especially the duration of the biological effects of a molecule according to the present disclosure.

A pharmaceutically acceptable carrier should not, by itself, induce antibodies deleterious to the individual receiving the composition and should not be toxic. Suitable carriers may be large slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and inactive virus particles.

Pharmaceutically acceptable salts can be used, for example, salts of minerals such as hydrochlorides, hydrobromides, phosphates and sulfates, or salts of organic acids such as acetates, propionates, malonates and Benzoates.

Pharmaceutically acceptable carriers in therapeutic compositions may additionally comprise liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, or pH buffering substances, can be present in such compositions. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions for ingestion by the patient.

Suitable forms for administration include those suitable for parenteral administration (eg, by injection or infusion, eg by bolus injection or continuous infusion). Where the product is for injection or infusion, it may take the form of a suspension, solution or emulsion in oily or aqueous vehicles and it may contain formulatory agents such as suspending agents, preservatives, stabilizers and/or Dispersant. Alternatively, antibody molecules may be in dry form for reconstitution with appropriate sterile fluids prior to use.

After formulation, the compositions of the invention can be administered directly to a subject. The subject to be treated can be an animal. However, in one or more embodiments, the compositions are adapted for administration to human subjects.

Suitably, in formulations of the present disclosure, the pH of the final formulation is not at a value similar to the isoelectric point of the antibody or fragment, for example, if the pH of the formulation is 7, a pi of 8-9 or above may be appropriate. While not wishing to be bound by theory, it is believed that this may ultimately provide a final formulation with improved stability, eg, the antibody or fragment remains in solution.

In one example, the pharmaceutical formulation at a pH in the range of 4.0 to 7.0 comprises: 1 to 200 mg/mL of an antibody of the disclosure, 1 to 100 mM buffer, 0.001 to 1% surfactant, a) 10 to 500 mM stabilizer, b) 10 to 500 mM stabilizer and 5 to 500 mM tonicity, or c) 5 to 500 mM tonicity.

The pharmaceutical compositions of the present invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, transdermal (see, for example, WO98/20734 ), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal, or rectal routes. The pharmaceutical compositions of the invention may also be administered using a hypodermic syringe. Typically, therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.

Direct delivery of such compositions will typically be achieved by injection subcutaneously, intraperitoneally, intravenously or intramuscularly or into the interstitial space of the tissue. Compositions can also be administered into lesions. Dosage therapy can be a single dose schedule or a multiple dose schedule.

It will be understood that the active ingredient in the composition will be the antibody molecule. Therefore, it will readily degrade in the gastrointestinal tract. Thus, if the composition is to be administered via the gastrointestinal route, the composition will need to contain an agent that protects the antibody from degradation but releases the antibody after it has been absorbed from the gastrointestinal tract.

A comprehensive discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Publishing Company, N.J. 1991).

In one embodiment, formulations are provided for topical administration, including formulations for inhalation.

Suitable inhalable formulations include inhalable powders, metered dose aerosols containing propellant gas or inhalable solutions without propellant gas. The active substance-containing inhalable powders of the present disclosure may consist of the above-mentioned active substance only or of a mixture of the above-mentioned active substance with physiologically acceptable excipients.

These inhalable powders may include monosaccharides (e.g. glucose or arabinose), disaccharides (e.g. lactose, sucrose, maltose), oligo- and polysaccharides (e.g. dextran), polyols (e.g. sorbitol, mannitol, wood sugar alcohols), salts (such as sodium chloride, calcium carbonate), or mixtures of these with each other. Suitably mono- or disaccharides are used, lactose or glucose is used, particularly but not exclusively in their hydrated form.

Particles for deposition in the lung need to have a particle size of less than 10 microns, such as 1 to 9 microns, eg 0.1 to 5 μm, specifically 1 to 5 μm. The particle size of the active ingredient (such as an antibody or fragment) is of primary importance.

Propellant gases useful in the preparation of inhalable aerosols are known in the art. Suitable propellant gases are selected from chlorinated and/or fluorinated derivatives of hydrocarbons such as n-propane, n-butane or isobutane and halogenated hydrocarbons such as methane, ethane, propane, butane, cyclopropane or cyclobutane things. The above-mentioned propellant gases may be used independently or in the form of mixtures thereof.

Particularly suitable propellant gases are halogenated alkane derivatives selected from TG11, TG12, TG134a and TG227. Among the aforementioned halogenated hydrocarbons, TG134a (1,1,1,2-tetrafluoroethane) and TG227 (1,1,1,2,3,3,3-heptafluoropropane) and mixtures thereof are particularly suitable.

Inhalable aerosols containing propellant gas may also contain other ingredients such as co-solvents, stabilizers, surface active agents (surfactants), antioxidants, lubricants, and means for adjusting the pH. All these ingredients are known in the art.

The propellant gas-containing inhalable aerosols according to the invention may contain up to 5% by weight of active substance. The aerosol formulations according to the invention comprise, for example, 0.002 to 5% by weight, 0.01 to 3% by weight, 0.015 to 2% by weight, 0.1 to 2% by weight, 0.5 to 2% by weight or 0.5 to 1% by weight of active ingredient.

Alternatively, topical administration to the lungs can also be achieved by, for example, using a device such as a nebulizer (e.g., a nebulizer connected to a compressor (e.g., a PariLC- JetPlus(R) sprayer)) to apply liquid solution or suspension formulations.

Antibodies of the invention may be dispersed in a solvent, eg, delivered as a solution or suspension. It can be suspended in a suitable physiological solution (eg, saline) or other pharmacologically acceptable solvent or buffer solution. Buffer solutions known in the art may contain 0.05 mg to 0.15 mg disodium edetate, 8.0 mg to 9.0 mg NaCl, 0.15 mg to 0.25 mg polysorbate, 0.25 mg to 0.30 mg anhydrous Citric acid, and 0.45 mg to 0.55 mg sodium citrate to achieve a pH of about 4.0 to 5.0. Suspensions can use, for example, lyophilized antibodies.

Therapeutic suspension or solution formulations may also contain one or more excipients. Excipients are well known in the art and include buffers (eg, citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohol, ascorbic acid, phospholipids, proteins ( For example, serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. Solutions or suspensions can be entrapped in liposomes or biodegradable microspheres. The formulations will generally be presented in substantially sterile form utilizing sterile manufacturing techniques.

This may include preparation and sterilization by filtering the buffered solvent/solution used for the formulation, aseptically suspending the antibody in a sterile buffered solvent solution, and dissolving the antibody by methods well known to those skilled in the art. The formulation is dispensed into sterile containers.

The sprayable formulations of the present disclosure can be presented, for example, as single-dose units enclosed in a foil-sealed pouch (eg, a sealed plastic container or vial). Each vial contains a unit dose in a solvent/solution buffer volume (eg, 2 mL).

Antibodies disclosed herein may be suitable for delivery by nebulization.

It is also contemplated that the antibodies of the invention may be administered through the use of gene therapy. To achieve this, DNA sequences encoding the heavy and light chains of the antibody molecule under the control of appropriate DNA components are introduced into the patient such that the antibody chains are expressed and assembled in situ from the DNA sequences.

In one embodiment, the present disclosure includes the use of an antibody or fragment thereof as a reagent or diagnostic agent, for example conjugated to a reporter molecule. A labeled antibody or fragment of the disclosure is thus provided. In one aspect, a column comprising an antibody or fragment of the disclosure is provided.

Accordingly, there is provided an anti-CSF-1R antibody or fragment for use as a reagent for:

1) Purification of CSF-1R protein (or binding fragment thereof) - which is conjugated to a matrix and used as an affinity column or (in a modified form of anti-CSF-1R) as a precipitant (e.g., to utilize Can be a modified form of the recognized domain) that is optionally precipitated by an anti-Fc reagent

2) Detection and/or quantification of CSF-1R on or in live or fixed cells (in vitro or in tissue or cell sections). Uses for this may include quantification of CSF-IR as a biomarker to follow the effect of anti-CSF-IR therapy. For these purposes, candidates can be used in a modified form (eg, by adding another moiety, as a gene fusion protein or a chemical conjugate, such as adding a reporter molecule, eg, a fluorescent tag for detection purposes).

3) Purification or sorting of CSF-1R-bearing cells labeled by binding to candidates modified in the manner exemplified in (1) and (2).

Comprising in the context of this specification means comprising.

Embodiments of the invention may be combined where technically appropriate.

Embodiments are described herein as including certain features/elements. The disclosure also extends to separate embodiments consisting or consisting essentially of said features/elements.

Technical references such as patents and applications are incorporated herein by reference.

Any embodiment specifically and expressly described herein may form the basis for a disclaimer either alone or in combination with one or more other embodiments.

Description of drawings

The invention is further described, by way of illustration only, in the following examples, which refer to the following drawings:

Figures 1A to 1F show certain amino acid and polynucleotide sequences.

Figures 2A to 2B show an alignment of certain sequences.

Figure 3 shows the sequence of the extracellular domain of human CSF-1R encoded by the polynucleotide used to transfect cells and express the protein on the cell surface. These cells are then used to immunize host animals.

Figure 4 shows inhibition of CSF-1 binding to THP-1 cells by antibody Ab969.

Figure 5a shows inhibition of CSF-1 driven survival and proliferation of primary human monocytes by antibody Ab969.

Figure 5b shows inhibition of IL-34-driven survival and proliferation of primary human monocytes by antibody Ab969.

Figure 6 shows inhibition of CSF-1 -driven survival and proliferation of primary murine monocytes by antibody Ab535.

Figure 7 shows the levels of cell surface CSF-1R on THP-1 cells incubated with Ab969, human CSF-1 and an isotype control.

Figure 8 shows the relative levels of cell surface CSF-1R on THP-1 cells treated with Ab969 compared to isotype controls.

Figure 9 shows the levels of cell surface CSF-1R on RAW264.7 cells incubated with Ab535, CSF-1 and an isotype control.

Figure 10 shows relative levels of cell surface CSF-IR on RAW264.7 cells treated with Ab535 compared to isotype control.

Figure 11 shows the level of CSF-1R phosphorylation on HEK293F cells transfected with CSF-1R after stimulation with CSF-1 or treatment with Ab969.

Figure 12 shows MCP-1 secretion levels of primary human monocytes incubated with Ab969 and with CSF-1.

Figure 13 shows the effect of inhibiting CSF-1 mediated monocyte survival by humanized antibody 969.g5 compared to chimeric Ab969.g0.

Figure 14 shows the effect of inhibiting CSF-1 mediated monocyte survival by various humanized antibody grafts of Ab969.

Figure 15a shows CSF-1 concentrations in serum samples collected from cynomolgus monkeys treated with a single iv dose of 7 mg/kg Ab969.g2.

Figure 15b shows CSF-1 concentrations in serum samples collected from cynomolgus monkeys treated with a single iv dose of 1.5 mg/kg Ab969.g2.

Figure 15c shows the effect of antibody Ab969.g2 administered to cynomolgus monkeys at doses of 7 mg/kg and 1.5 mg/kg on circulating atypical monocytes at different time points.

Figure 16 shows the anti-tumor effect of antibody Ab535 relative to control antibody, positive control and vehicle control in immunodeficient nude mice bearing subcutaneous grafts of human breast cancer xenograft MCF-7.

Figure 17 shows the anti-tumor efficacy of antibody Ab535 in orthotopic prostate cancer model PC-3 relative to control antibody, positive control and vehicle control.

Figure 18a shows the effect of Ab535 treatment on bleomycin-induced lung fibrosis on BALF collagen concentration compared to isotype control.

Figure 18b shows the effect of Ab535 treatment of bleomycin-induced lung fibrosis on fibrotic pathology in lung samples as measured by the Ischler score compared to isotype control.

Figure 18c shows the effect of Ab535 treatment on bleomycin-induced lung fibrosis on albumin concentration in serum compared to isotype control.

Figure 18d is the number of macrophages in the BAL fluid of mice from the Bleomycin-induced pulmonary fibrosis study and shows the effect of Ab535 on Bleomycin-induced pulmonary fibrosis compared to isotype control treated animals The treatment of BAL decreased the number of macrophages in the BAL fluid. Data are shown as mean ± SEM; * indicates significantly different from saline isotype; # indicates significantly different from bleomycin-treated mice administered with isotype control antibody (p<0.05)

Figure 19 shows representative images of histopathological analysis of lungs from saline control, bleomycin+isotype control, and bleomycin+Ab535 treated animals.

Figure 20a shows the effect of Ab535 treatment of ADA deficient mice with induced lung fibrosis on BALF collagen concentration compared to treatment with isotype control.

Figure 20b shows the effect of Ab535 treatment of ADA-deficient mice with induced lung fibrosis on the fibrotic pathology of lung samples as measured by the Ehrlich score compared to treatment with an isotype control.

Figure 20c shows the effect of Ab535 treatment of ADA deficient mice with induced lung fibrosis on BALF collagen concentration compared to treatment with isotype control.

Figure 20d shows the number of macrophages in the BAL fluid of mice from an ADA-deficient model of pulmonary fibrosis and shows that Ab535 treatment of ADA-deficient mice with induced pulmonary fibrosis reduces the number of macrophages in the BAL fluid .

Figure 21 shows representative images from histopathological analysis of lungs from normal mice (ADA+) and ADA-deficient mice (ADA-) with induced pulmonary fibrosis treated with isotype control or Ab535.

Example

Example 1 - Production of Anti-CSF-1R Antibodies

Immunization:

Female Sprague Dawley rats were immunized with syngeneic RFL6 rat fibroblasts transiently transfected with a vector expressing the extracellular domain of human CSF-1R linked to a glycosylated phosphatidylinositol (GPI) anchor. Figure 3 shows SEQ ID NO: 39, which is the human CSF-1R ectodomain sequence used for immunization of rats.

Rats received five subcutaneous immunizations of ^3-9x106 transfected cells per animal at three week intervals. Freund's complete adjuvant (50% in PBS) was injected adjacent to the site of the first cell immunization. Two weeks after the last immunization, peripheral blood mononuclear cells (PBMC) and spleens were harvested.

Primary Screening of Antibody Supernatants

Antibody supernatants were first screened for their ability to bind to human CSF-IR expressed on transfected HEK293 cells by Fluorescence Microassay Technology (FMAT).

Approximately 1000 wells with anti-CSF-1R reactivity were identified in the primary FMAT screen of 600x96 well plates. A total of approximately ^3x108 B cells (rat splenocytes) were screened by FMAT for the production of CSF-1R binding antibodies.

Secondary Screening of Antibody Supernatants

A medium-throughput assay has been designed to identify FMAT-positive wells containing neutralizing anti-CSF-1R activity (ie, antibodies with the ability to prevent human CSF-1 from binding to the human CSF-1R receptor). Antibody supernatants were incubated with THP-1 cells expressing CSF-1R prior to incubation with human CSF-1. The level of CSF-1 bound to THP-1 cells was measured by flow cytometry using an anti-CSF-1 polyclonal antibody. Use an irrelevant antibody supernatant as a negative control.

A secondary screen was applied to supernatants from 779 antibody wells and 88 of these wells exhibited detectable CSF-1 blocking activity.

Tertiary screening of antibody supernatants

Antibody supernatants that had shown neutralizing activity in the secondary screen were tested for their ability to prevent CSF-1 dependent survival of primary human monocytes. Monocytes were purified from human blood and ¹ x 104 cells were incubated with each antibody supernatant in the presence of 20 ng/ml human CSF-1. After 72 hours of incubation, the number of viable monocytes was measured by the CellTiterGlo assay.

The tertiary screen was applied to supernatants from 59 antibody wells identified in the secondary screen and 18 wells exhibited the ability to reduce CSF-1 -mediated monocyte survival.

Cloning of antibody variable regions and reanalysis of blocking activity

From these 18 antibody wells variable region (V-region) cloning was attempted to confirm CSF-1 neutralizing activity. Heavy and light chain immunoglobulin V-regions were amplified by RT-PCR using primers specific for rat antibody constant regions and a redundant primer set annealing to a sequence encoding the rat immunoglobulin leader peptide. V-region genes were recovered from 14 antibodies and cloned into heavy and light chain human IgG4 expression vectors.

Antibody vector pairs were transiently transfected into HEK293F cells and conditioned media (as described above) were tested for CSF-1 neutralizing activity on THP-1 cells. Nine of the antibodies had the ability to partially or completely inhibit CSF-1 binding compared to the control conditioned medium.

Sequence Analysis of Nine Neutralizing Anti-CSF-1R Antibodies

Nine neutralizing antibodies were sequenced to assess sequence diversity. All nine of these antibodies are unique. These antibodies were cloned and expressed for further characterization studies.

Example 2 - In vitro properties of anti-human CSF-1R chimeric Ab969 and anti-mouse CSF-1RAb535

i) Expression of chimeric Ab969

The variable regions of the nine neutralizing antibodies identified in Example 1 were cloned into separate heavy and light chain expression vectors and expressed as full-length human IgG4 antibodies.

The VH gene was cloned into the vector pVhg4FL(V19H) containing DNA encoding the native leader sequence and the human gamma-4 heavy chain constant region with the hinge stabilizing mutation S241P. The VL gene (κ) was cloned into the vector pKH10.1 (V4L) containing DNA encoding the native leader sequence and the human κ chain constant region (Km3 allotype).

Antibodies were expressed by transient co-transfection of matching heavy and light chain vector pairs into CHO-K1 cells. Antibody purification in PBS pH 7.4 was performed such that the concentration of aggregates in the final preparation was less than 1%.

The panel of nine antibodies contained the antibody designated Antibody 969. A chimeric antibody comprising a rat variable region and a human γ-4 heavy chain constant region and a human κ chain constant region is referred to as Antibody 969 or Antibody 969cHcL or Antibody 969.g0 in the Examples below.

ii) Ligand Blocking Assay

The ability of each of these nine antibodies to inhibit CSF-1 binding to THP-1 cells was assessed by flow cytometry. THP-1 cells were incubated with 0.5, 0.125, 0.031, 0.0078 and 0.00195 μg/ml of each antibody for 30 minutes. An irrelevant IgG4 antibody served as an isotype control. After washing, cells were incubated with 0.5 μg/ml human CSF-1 for 30 minutes. After further washing, bound CSF-1 was detected by sequential incubations with biotinylated anti-CSF-1 antibody and Alexa488-conjugated streptavidin. Ligand binding to the receptor was measured by flow cytometry and median fluorescence intensity (MFI) was plotted.

This assay identified four antibodies (including Ab969) superior to the remaining antibodies tested, demonstrating complete inhibition of CSF-1 binding at a concentration of 31.3 ng/ml. The results for Ab969 are shown in FIG. 4 .

iii) CSF-1- and IL-34-mediated inhibition of monocyte survival

Anti-human CSF-1R:

Purified anti-human CSF-1R antibodies were tested for their ability to inhibit CSF-1 and IL-34 driven survival and proliferation of primary human monocytes. In each assay, mitogens (CSF-1 or IL-34) were used at concentrations that provided maximal stimulation of monocytes.

Human PBMC were prepared from fresh human whole blood on a Ficoll gradient and monocytes were purified by negative selection. Monocytes were incubated with 0.25 μg/ml antibody and 20 ng/ml CSF-1 for 72 hours and the relative number of viable cells was determined using CellTiterGlo assay. Luminescence readouts correlate with the number of viable cells. The results are shown in Figure 5a.

Human PBMC were prepared from fresh human whole blood on a Ficoll gradient and monocytes were purified by negative selection. Monocytes were incubated with 0.25 μg/ml antibody and 20 ng/ml IL-34 for 72 hours and the relative number of viable cells was determined using CellTiterGlo assay. Luminescence readouts correlate with the number of viable cells. The results are shown in Figure 5b.

These four antibodies, including Ab969, showed superior inhibition of monocyte survival on CSF-1 (Figure 5a) and IL-34 (Figure 5b) stimulation compared to the remaining antibodies, consistent with their ligand blocking activity.

Anti-mouse CSF-1R:

Purification of primary murine CD11b ⁺ monocytes from mouse spleens. Monocytes were then incubated for 24 hours with a titration of murine CSF-1 (mCSF-1). The release of MCP-1 into the cell culture medium was measured by ELISA and a dose-dependent release of MCP-1 by mCSF-1 was confirmed. In a separate group in this study, anti-mouse CSF-1 RAb535 was added at 10 μg/ml with mCSF-1 titration. Ab535 completely inhibited the release of MCP-1 at all concentrations of CSF-1 tested (Figure 6).

MCP-1 release assays were performed using primary murine monocytes at a constant CSF-1 concentration of 100 ng/ml and titration of Ab535. Dose-dependent inhibition of MCP-1 release was confirmed and _IC50 calculated. The average _IC50 of Ab535 from two independent experiments was determined to be 8.08 ng/ml.

Conclusions: Treatment of murine monocytes with CSF-1 resulted in a dose-dependent release of MCP-1 which was completely inhibited by treatment with 10 μg/ml Ab535. Ab535 inhibited CSF-1 -driven MCP-1 release from murine monocytes in a dose-dependent manner with a mean _IC50 of 8.08 ng/ml (n=2). This _IC50 value is similar to that exhibited _by anti-human CSF-1R antibody.

iv) Affinity

The ability of antibodies to bind CSF-1R was tested in a BIAcore assay by measuring the binding kinetics against purified recombinant CSF-1R/Fc fusion protein.

The assay format was capture of anti-CSF-1R antibody by immobilized anti-human IgG, F(ab') ₂ followed by titration of hCSF-1R/Fc on the capture surface. BIA (Biamolecular Interaction Analysis) was performed using BIAcore3000 (GE Healthcare Bio-Sciences AB). All experiments were performed at 25°C. Specific Affinipure F(ab') ₂ fragment goat anti-human IgG, F(ab') ₂ fragment (Jackson ImmunoResearch) was immobilized via amine coupling chemistry on CM5SensorChip (GE Healthcare Bio-Sciences AB) to a level of ~5000 response units (RU) . HBS-EP buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 m MEDTA, 0.005% surfactant P20, GE Healthcare Bio-Sciences AB) was used as running buffer at a flow rate of 10 μl/min. Injection of anti-CSF-1R antibody was performed to generate a capture level of approximately 100 RU on immobilized anti-human IgG, F(ab) ₂ .

Recombinant human CSF-1R/Fc (R&D Systems) was passed over the captured anti-CSF-1R antibody at 5nM at a flow rate of 30ul/min for 5min, then the flow rate was increased to 100ul/min for 30min during the dissociation phase. Injections at 5 nM were performed twice along with corresponding buffer controls. These sensorgrams are used to generate off-rates. Recombinant human CSF-1R/Fc was titrated on captured anti-CSF-1R antibody from 2.5nM at a flow rate of 30ul/min for 5min, followed by a 10min dissociation phase. These sensorgrams were used to generate association rates. The surface was regenerated by a 10 ul injection of 40 mM HCl followed by a 5 ul injection of 10 mM NaOH at a flow rate of 10 ul/min. Double-referenced background-subtracted binding curves were analyzed following standard procedures using BIA Evaluation software (version 4.1). Kinetic parameters were determined from the fitting algorithm.

Three of the four tested antibodies exhibited affinities (K _D ) of less than 10 pM. Advantageously, this is compared with the anti-mouse CSF-1R parallel reagent Ab535. The results for Ab969 and Ab553 are shown in Table 1.

Table 1:

Antibody affinity was also measured by cell-based assays using THP-1 cells. Antibodies were directly labeled using Alexa-488 fluorescent dye and affinity was measured by quantitative flow cytometry. Additional BIAcore analysis confirmed that fluorescent conjugation of the antibody did not alter the affinity for recombinant CSF-1R protein. The results are shown in Table 2.

The absolute affinity values obtained by the two methods are different, and this difference is often observed when comparing the two systems. However, cell-based methods demonstrated that antibodies bind CSF-1R with high affinity.

Table 2

v) Inhibition of CSF-1 binding ( _IC50 )

Four antibodies were analyzed by measuring their relative potency to inhibit CSF-1 binding to THP-1 cells. All four antibodies exhibited potent inhibition of CSF-1 binding in this assay format with _IC50 values of less than 5 ng/ml (-30 pM).

table 3

Antibody Mean IC ₅₀ ±SD(ng/ml) Ab969 2.89±1.22

vi) Antibody cross-reactivity

Four antibodies were tested for cross-reactivity with rhesus monkey, cynomolgus monkey, and canine full-length CSF-1R. In addition to human CSF-1R, all four antibodies also bound to rhesus and cynomolgus CSF-1R, showing significant binding significantly above isotype control levels.

Table 4

vii) CSF-1R Internalization

Ab969:

THP-1 cells were incubated with Ab969 for 0, 0.5, 2, 4, 24 and 48 hours. Cells were also treated with human CSF-1 and an isotype control, which served as positive and negative controls, respectively. At each time point, cell surface CSF-IR levels were measured by flow cytometry (Figure 7). Relative levels of cell surface CSF-1R on cells treated with Ab969 relative to the isotype control were calculated and shown in FIG. 8 .

Treatment of THP-1 cells with recombinant CSF-1 results in a rapid and sustained decrease in cell surface CSF-1R levels; the native ligand binds to its cognate receptor and drives internalization of the ligand-receptor complex.

THP-1 cells treated with Ab969 exhibited higher levels of cell surface CSF-1R expression throughout the 48 hour time course compared to untreated and isotype control treated THP-1 cells. This data strongly suggests that Ab969 treatment of THP-1 cells does not result in internalization of cell surface expressed hCSF-1R up to 48 hours after treatment.

Cell surface expression of CSF-1R was significantly increased over time on untreated and treated THP-1 cells, except for cells treated with CSF-1. This could be due to expression changes during cell growth over the 48 hour period of the experiment and could potentially be a stress response.

To provide further evidence that Ab969 does not enhance receptor internalization, and also to rule out the possibility that the THP-1 cell line is not physiologically relevant, primary human monocytes were also used in this internalization. Data from this assay also demonstrates that Ab969 does not result in rapid internalization of cell surface CSF-IR on healthy primary human monocytes.

Ab535:

The mouse leukemia monocyte/macrophage cell line RAW264.7 expresses high levels of murine CSF-1R (mCSF-1R) and represents a suitable candidate for testing whether the anti-mouse CSF-1R antibody Ab535 can cause receptor internalization cell-based system.

RAW264.7 cells were incubated with Ab535 for 0, 0.5, 2, 4, 24 and 48 hours. Cells were also treated with human CSF-1 and an isotype control as positive and negative controls, respectively. At each time point, cell surface CSF-1R levels were measured by flow cytometry (Figure 9). Relative levels of cell surface CSF-1R relative to isotype control were calculated and shown in FIG. 10 .

The data show that treatment of THP-1 cells with recombinant CSF-1 results in a rapid and sustained decrease in cell surface CSF-1R levels.

A clear reduction in cell surface mCSF-1R levels relative to untreated and isotype control treated cells was observed on RAW264.7 cells treated with human CSF-1 for 2 hours. This reduction was maintained throughout the 48 hour study. This confirms that human CSF-1 causes internalization of mCSF-1R and validates the experimental system used to monitor receptor internalization.

RAW264.7 cells treated with Ab535 exhibited similar levels of cell surface mCSF-1R throughout the 48 hour time course compared to untreated and isotype control treated cells. Because antibody-mediated receptor internalization is expected to occur within this time frame, the data strongly suggest that Ab535 does not trigger mCSF-IR internalization.

To provide further evidence that Ab535 does not enhance receptor internalization, primary murine CD11b ⁺ monocyte-macrophages were used in the internalization assay. These results also demonstrate that treatment of mouse monocyte-macrophage cells with Ab535 did not result in internalization of cell surface expressed mCSF-IR up to 24 hours after treatment.

viii) CSF1-R activation

Binding of CSF-1 to CSF-1R leads to the formation of receptor dimers, which triggers rapid receptor phosphorylation by bringing the kinase domains into close proximity. This subsequently leads to receptor internalization and activation of several well-characterized signaling pathways, including the Ras-MAPK pathway. It is possible that anti-CSF-1R antibodies could cause receptor clustering and trigger downstream signaling cascades. This may be an undesirable property of an antibody that is supposed to inhibit receptor signaling, therefore, Ab969 was tested for receptor agonism using two independent in vitro assay formats.

In the first assay format, antibodies were incubated with cells transfected with human full-length CSF-IR and phosphorylation of downstream signaling molecules as well as receptors were monitored by Western blotting.

The THP-1 cell line represents a starting point for monitoring the activation status of CSF-1R. However, the expression level of CSF-1R in this cell line is rather low and biochemical analysis is difficult. Therefore, the experimental system was designed to robustly detect the level of CSF-1R phosphorylation. Phosphorylation of CSF-1R can be detected on two tyrosine residues, Y723 and Y809. In addition, phosphorylation of p44/42MAPK (Erk1/2) on T202 and Y204 was measured as an independent readout of CSF-1R activity. In all experiments, stimulation with CSF-1 was included as a positive control.

HEK293F cells were transfected with a plasmid vector expressing full-length CSF-1R. After 24 hours of incubation in serum-free conditions, cells were stimulated with a 100 μg/ml to 0.001 μg/ml titration of Ab969.g0 for 5 minutes. Cells were also treated with 500ng/ml recombinant CSF-1 to provide a positive control. Include untreated cells as a negative control. Protein lysates from treated and untreated cells were separated by SDS-polyacrylamide electrophoresis and blotted onto nitrocellulose. Using anti-phospho-Y723CSF-1R (CellSignalingTechnology #3151), phospho-Y809 (CellSignalingTechnology #3154), total CSF-1R (CellSignalingTechnology #3152) and phospho-ERK1/2 (p44/42MAPK) (CellSignalingTechnology #5301) antibody for Western blotting.

Unstimulated CSF-1R transfected HEK293F cells exhibited low basal levels of CSF-1R phosphorylation. Stimulation of cells with CSF-1 resulted in phosphorylation levels of CSF-1R on residues Y723 and Y809, which were readily visualized by Western blot analysis (Figure 11). There was also a clear stimulation of ERK1/2 phosphorylation following CSF-1 treatment. Treatment of transfected HEK293F cells with antibody Ab969.g0 did not stimulate phosphorylation of CSF-1R or enhance ERK1/2 activation.

In a second assay, antibody Ab969 (monomeric and cross-linked) was incubated with primary human monocytes and MCP-1 secretion was used as a marker of CSF-1R activation. CSF-1 treatment of human monocytes causes them to release monocyte chemoattractant protein-1 (MCP-1). MCP-1 release would be expected to occur if anti-CSF-1R antibodies had the ability to activate the CSF-1R receptor on monocytes.

The previously described biochemical assays used only monomeric anti-CSF-1R antibodies. However, it is possible that cross-linked IgG1 would have an enhanced ability to bring CSF-1R molecules into close proximity and trigger tyrosine phosphorylation and downstream signaling. To assess this, an MCP-1 assay was also performed using Ab969.g0 that had been cross-linked with an anti-human Fc antibody.

Human monocytes can be prepared from human whole blood as follows: Collect 60 to 100 ml of human whole blood in BD Vacutainer 10 ml Lithium Heparin 171 IU tubes. Blood was divided into 3-4 Leucosep Ficoll tubes (GreinerBio-One) and topped up with PBS. The PBMC layer was collected by centrifuging the tubes without interruption at 1000 g for 10 minutes at 20°C. Cells were pelleted and monocytes were subsequently isolated using positive selection human CD14 beads (Miltenyi Biotec 130-050-201 ) according to the manufacturer's protocol. Antibody Ab969.g0 was cross-linked by adding goat anti-human IgG Fc antibody (R&D Systems G-102-C) at a ratio of Ab969.g0:Fc of 2:1. Monocytes were seeded at 20,000 cells/well in culture in the presence of antibody Ab969.g0 or cross-linked Ab969.g0 in a dose titration (semi-log serial dilution, including 16 concentrations, with a maximum concentration of 10 μg/ml) Base. Control wells contained no Antibody. The cells were incubated for 24 hours, the plates were centrifuged to pellet the cells, and the supernatant collected. Secreted MCP-1 was measured by MSD (K151AYB-2) according to the manufacturer's protocol.

The results from the experiments are shown in FIG. 12 . No increase in MCP-1 levels was detected for any Ab969.g0 treatment, whether in monomeric or cross-linked form; the concentration of MCP-1 in the culture medium of treated cells was the same as that of untreated cells. As expected, cells treated with CSF-1 resulted in a significant and reproducible increase in MCP-1 levels.

Example 3 - Humanization of Antibody 969 and Selection of Humanized Grafts

Four anti-CSF-1R antibodies were selected for humanization based on their affinities and properties measured in Example 2.

i) Generation of humanized grafts

Antibodies 969 and 970 were humanized by grafting CDRs from the rat antibody V region onto the human germline antibody V region framework.

The CDRs grafted from the donor to the recipient sequence are as defined by Kabat (Kabat et al., 1987) except for CDR-H1 where the combined Chothia/Kabat definition is used (see Adair et al., 1991 Humanised antibodies. WO91/09967) .

The human V region 2-1-(1)O12+JK4J-region (VBASE, http://vbase.mrc-cpe.cam.ac.uk/) was chosen as the acceptor for the light chain CDR. The human V region VH23-12-70+JH3J region (VBASE, http://vbase.mrc-cpe.cam.ac.uk/) was chosen as the acceptor for the heavy chain CDRs.

Many framework residues from the rat V region were retained in the humanized sequence, as shown in Table 1.

An automated synthetic approach was used to design and construct genes encoding the initial humanized light and heavy chain V region sequences (designated gL1 and gH1, respectively). Other variants of the light and heavy chain V regions were created by modifying the gL1 and gH1 genes with oligonucleotide site-directed mutagenesis.

The VK genes (gL1 to gL9) were cloned into the human light chain expression vector pKH10.1 containing DNA encoding the human kappa chain constant region (Km3 allotype). The VH genes (gH1 and gH2) were cloned into the human γ-4 heavy chain expression vector pVhγ4PFL containing DNA encoding the human γ-4 heavy chain constant region with the hinge stabilizing mutation S241P (Angal et al., 1993, Mol Immunol. 30: 105-8). Different combinations of plasmids encoding the variant light and heavy chains were co-transfected into HEK293F, resulting in the expression of humanized recombinant 969 antibodies.

The other three anti-CSF-1R antibodies also provided conserved grafts containing many donor residues predicted to be of importance in the heavy and light chains as well as fully humanized grafts containing no donor residues. source.

ii) Affinity of humanized antibody

Each humanized graft was assessed for (i) binding affinity to human CSF-IR as measured by BIAcore and (ii) melting temperature (Tm) as measured by ThermoFluor assay relative to the parental chimeric antibody. The melting temperature is believed to provide an early indication of the stability of the antibody molecule, with unstable antibodies typically exhibiting a Tm of less than 75.0°C.

Grafts representing the humanized stage of Ab969 are shown in Table 5. Table 5 also shows the chimeric antibody (969cHcL) of Ab696. The conserved graft ( _969gH1gL1 ) exhibited an affinity constant (KD) of 2.4 pM, thus no significant loss of affinity compared to the chimeric rat antibody (969cHcL). The Tm of the 969gH1gL1 conserved graft was 78.8°C and thus above the threshold of 75.0°C. Substitution of V78 by the A78 donor residue in the heavy chain to generate _969gH2gL1 did not reduce antibody affinity (KD = 2.3 pM). No change in affinity was observed after stepwise replacement of K38, Y71 and F87 donor residues to Q38, F71 and Y87, respectively. The final humanized graft, 969gH2gL8, which did not contain donor residues, exhibited similar affinity (4.1 PM) to the parental chimeric antibody.

table 5

Ab969 has a potential DG isomerization motif at the junction of CDR-L2 and the framework. This DG site aspartic acid residue in the 969gH2gL7 and 969gH2gL8 grafts was mutated to serine to provide the inotropic SG sequence. Affinity to CSF-IR was measured by BIAcore and no apparent loss of affinity was detected (Table 6). Furthermore, the final 969H2gL7(SG) and 969gH2gL8(SG) grafts retained high Tm of 80.5°C and 79.9°C, respectively.

Table 6

Humanization of Ab969 and one other anti-CSF-1R antibody, _Ab970 , resulted in a fully humanized antibody with an affinity (KD) comparable to the parental chimeric antibody and a Tm giving an initial prediction of molecular stability (absence of rat donor residue). The fully humanized form of Ab969 (969gH2gL8SG) was renamed Ab969.g5. Hereafter, the chimeric form of Ab969 will be referred to as Ab969.g0.

The affinity of purified chimeric and humanized grafts of the Ab969 antibody towards recombinant CSF-IR was again measured using BIAcore analysis. Previous BIAcore experiments performed during the humanization process were performed using crude cell supernatants instead of purified antibodies. _A slight decrease in affinity was detected, with an increase in KD value from about 4 pM to 5 pM.

Table 7

iii) Inhibition of CSF-1-mediated monocyte survival by Ab969.g0 and Ab969.g5

CSF-1 is essential for the activation and survival of cultured monocytes; if CSF-1 is removed, monocytes rapidly undergo apoptosis. An assay was designed using MCP-1 (Monocyte Chemoattractant Protein-1; also known as CCL2, Chemokine C-C Motif Ligand 2) as readout. Following CSF-1 stimulation, human monocytes secrete MCP-1, which can be detected by ELISA in cell supernatants usually at 24 hours post-stimulation. Inhibition of CSF-1R signaling by blocking CSF-1-binding antibodies results in a decrease in MCP-1 secretion.

Human PBMC were prepared from fresh human whole blood on a Ficoll gradient and CD14 ⁺ monocytes were purified by positive selection. ^A total of 2x104 monocytes were incubated with semi-log serial dilutions of antibody from 10 μg/ml to 0.35 pg/ml for 24 hours in the presence of 100 ng/ml human CSF-1. Controls containing "no antibody, no CSF-1" and "no antibody, with CSF-1" were included to provide minimum and maximum MCP-1 release values. After 24 hours of incubation, the cells were pelleted by centrifugation and the supernatant collected. The concentration of MCP-1 was measured using human CCL2/MCP-1 DuoSet ELISA (R&D Systems DY279) following the manufacturer's instructions.

Hereinafter, this assay is referred to as "MCP-1 inhibition assay".

MCP-1 inhibition assay was used to compare the activity of humanized 969.g5 and chimeric Ab969.g0. Five independent assays were performed using four different donors of monocytes. Ab969.g5 humanized grafts exhibited unexpectedly significantly lower activity compared to the parental chimeric antibody 969.g0 in all assays. A single representative experiment is shown in Figure 13. The mean _IC50 for 969.g0 in the monocyte assay was 24.6 ng/ml compared to 333.0 ng/ml for 969.g5. This indicates a 13.5-fold reduction in antibody potency when using this assay format to compare the two antibodies.

A series of experiments were performed using Ab969 to show why the humanized antibody exhibited reduced activity in the MCP-1 inhibition assay. The data show that the loss of Ab969.g5 activity observed in the MCP-1 inhibition assay is due to the sequence of addition of antibody and ligand to target cells. The activity of both Ab969.g0 and Ab969.g5 was reduced when the competition assay format was applied, but more importantly, a large difference in their blocking activity was detected. To analyze whether the lower "on-rate" of humanized Ab969 could be responsible for the reduced potency, BIAcore analysis was performed. These data identified that while the K _D of Ab969.g0 and Ab969.g5 were similar, the Ka of Ab969.g5 was lower at 1.57x10 ⁶ M compared to 2.16x10 ⁶ M ^-1 s ^-1 for 969.g0 ^-1 s ^-1 (see Table 5). In a competition assay, where CSF-1 and anti-CSF-1R antibodies compete for binding to the same receptor, slower antibody association rates if the association rate against the ligand is also high and the ligand is present at high concentrations May result in reduced blocking activity. Human CSF-1 is known to have a particularly high association rate of 22.19x10 ⁶ M ⁻¹ s ⁻¹ similar to antibodies, and the assay was performed at a high CSF-1 concentration (250 ng/ml).

To further confirm that the reduction in blocking activity of 969.g5 was due to the intrinsic properties of the antibody, an ELISA measuring the binding of CSF-1 to CSF-1R was developed. This method is hereinafter referred to as "ELISA ligand-blocking assay". This assay was performed using competitive binding, where CSF-1 and antibody were premixed prior to application to plate-bound CSF-1R. An assay was also performed to assess the effect of CSF-1 concentration on antibody activity.

When IC50s of 969.g0 and 969.g5 were measured using a CSF-1 concentration of 1 ng/ml in a ligand blocking ELISA, the two antibodies appeared to have an _IC50 of _12.83 ng/ml vs. 19.65 ng/ml, respectively similar activity. When a concentration of 10 ng/ml CSF-1 was used in the assay, the IC ₅₀ of both Ab969.g0 and Ab969.g5 increased, but more importantly, the difference between the two increased further to 79.29 ng/ml vs 268.10 ng/ml, respectively. ml. This trend continued in assays using 100 ng/ml CSF-1, where Ab969.g0 and Ab969.g5 gave _IC50 values of 828.70 ng/ml and 3947.00 ng/ml, respectively. Competition assays demonstrated that Ab969.g5 was less active than Ab969.g0 in blocking CSF-1 binding to CSF-1R. This decrease in potency became more pronounced with increasing CSF-1 concentrations.

iv) Identification of humanized grafts of Ab969 with comparable activity to chimeric Ab969 in MCP-1 inhibition assays

A panel of Ab969 humanized intermediate grafts was prepared by transient expression to allow comparable in vitro activities (Table 8). Corresponding chimeric antibodies (Ab969.g0) and fully humanized grafts (Ab969.g5) were also included in the transient expression to allow direct comparison of antibody characteristics within the same batch.

Table 8:

The in vitro activity of each antibody was tested in the MCP-1 inhibition assay. An assay format using CSF-1 titration with a single concentration of antibody was chosen as this format enabled rapid screening of large antibody panels and highlighted any differential CSF-1R blocking activity. In this assay, a dose-dependent release of MCP-1 from primary human monocytes was detected and the relative ability of each antibody to block CSF-1R activity was measured by the concentration of CSF-1 at which MCP-1 was released. Monocytes were seeded in culture medium at 20,000 cells/well in the presence of a titration of recombinant human CSF-1 (2-fold serial dilution including 18 concentrations up to a maximum concentration of 500 ng/ml). A single dose of 1 μg/ml of antibody was added. Cells were incubated for 24 h and supernatants were collected. Secreted MCP-1 was measured by ELISA. MCP-1 inhibition assays showed that antibodies Ab969.g2 and Ab969.g7 retained high CSF-1R blocking activity, where both entities were able to completely inhibit MCP-1R when CSF-1 was added to monocytes at concentrations greater than 100 ng/ml 1 secreted (Figure 14). In this assay, a significant loss of activity was detected for Ab969.g5, which could only inhibit MCP-1 secretion up to concentrations of 10 ng/ml CSF-1. Similarly, antibody Ab969.g9 exhibited reduced CSF-1R blocking activity compared to Ab969.g2 and Ab969.g7.

The _IC50 of selected Ab969 humanized grafts was measured in the MCP-1 assay to more thoroughly assess their relative ability to inhibit CSF-1 mediated monocyte activation. It is considered important to assess antibody activity using several different donors without mixing monocytes from mixed sources. Table 9 shows the _IC50 values of the antibodies accumulated from assays performed on Ab969 using 6 different donors.

Both Ab969.g2 and Ab969.g7 humanized grafts showed relative IC50s comparable to the chimeric _Ab969.g0 antibody. In marked contrast, Ab969.g5 exhibited much less potency in the assay (average chimera/graft _IC50 ratio of 19.6).

Table 9:

v) Affinity of the humanized Ab696 antibody panel

The affinity of each Ab969 humanized antibody graft and the parental chimeric antibody was measured by BIAcore (Table 10), where three independent experiments were performed and the mean value calculated. The data show that the affinity (K _D ) does not appear to change during humanization from the chimeric molecule (Ab969.g0) to the "fully humanized" antibody Ab969.g5. However, the antibody "association rate" decreased as humanization proceeded beyond the Ab969.g2 graft; both Ab969.g4 and _Ab969.g5 had lower Ka values than the previous graft. In addition, the Ka for _Ab969.g5 was lower than for Ab969.g4, potentially suggesting that mutation of the DG isomerization site to SG further reduces the antibody association rate.

Table 10:

in conclusion:

Testing of groups of Ab969 humanized grafts in several assays showed that a tyrosine residue at position 71 in the light chain (eg Y71 ) improved the activity of the antibody. Substitution of, for example, phenylalanine by Y71 results in _a decrease in antibody association rate (decreased Ka) relative to the parental chimeric molecule. This resulted in a reduced ability of the antibody to block the binding of CSF-1 to CSF-1R, as shown in an assay monitoring the activity of CSF-1R in primary human monocytes (MCP-1 inhibition assay).

Example 4 - Molecular stability of the humanized Ab696 panel

i) thermal stability

Thermal stability (measured as melting temperature, Tm) was determined by two independent methods; one by monitoring unfolding by binding a fluorescent dye to an exposed hydrophobic surface (Thermofluor method), the other by calorimetric analysis (DSC), which is an orthogonal technique.

_Tm measured by Thermofluor against antibody 969 (in PBS pH7.4) for various grafts is summarized in Table 11.

Table 11

Overall, Fab'T _m as measured by Thermofluor showed that most 969 grafts were more thermostable compared to IgG4 controls.

ii) Effect of sample concentration on aggregation propensity

As a predictor of sample stability during storage, the effect of antibody concentration on stability in PBS pH 7.4 was studied.

Experiment 1:

Antibodies were concentrated to >10 mg/ml and incubated at room temperature for 5 days. Immediately after concentration (T ₀ ), 969.g7 appeared cloudy and 969.g8 appeared slightly opalescent. In contrast, the 969.g5 sample was judged to be clear by visual inspection. After 5 days of incubation at room temperature, the 965.g5 sample remained clear, while the aggregation of 969.g7 and 969.g8 had developed further, each showing a large amount of precipitate.

From this study the samples can be ranked in order of resistance to aggregation under these conditions as follows: 969.g5>969.g8>969g7.

Experiment 2:

Prior to concentration, all three antibody samples were observed to be clear by visual inspection, except for 969.g6 (4.68 mg/ml), which appeared opalescent. After concentration to 16 mg/ml, a precipitate of 969.g6 was observed both at room temperature and after 24 hours of storage at 4°C. However, both 969.g1 and 969.g4 remained visually clear. After 5 days of further incubation, apparently large particles were observed for sample 969.g1 at both room temperature and 4°C. No aggregation of sample 969.g4 was observed at room temperature or 4°C as judged by visual inspection.

From this study it can be shown that 969.g6 has some aggregation instability when stored at low concentration (4.68 mg/ml) in PBS pH 7.4. Furthermore, this precipitation is exacerbated by further antibody concentration. Concentrated 969.g1 showed a slower rate of aggregation, while concentrated 969.g4 remained stable for up to 5 days at either storage temperature. The order of stability against aggregation is thus: 969.g4>969.g1>969.g6.

Experiment 3: Antibodies 969.g2, 969.g5, 969.g7 and 969.g9

Samples were analyzed immediately after concentration (T ₀ ), after overnight incubation and at 5 days after concentration. All samples were observed to be clear and there was no evidence of particle formation based on visual inspection prior to concentration. Immediately after concentration to 23.07 mg/ml, the 969.g7 sample showed precipitation as previously observed in Experiment 1 . A slight opalescence was observed for 969.g9, which became more pronounced after 24 hours storage at room temperature or 4°C. All other 969 grafts were observed to appear clear upon visual inspection immediately after enrichment.

After 21 days of incubation, there were no further visible changes compared to that observed after overnight incubation at room temperature; both 969.g7 and 969.g9 grafts aggregated due to concentration of the samples, however, the other samples did not Aggregation was clearly visible.

Overall, the 969.g2 sample showed minimal tendency to aggregate due to increasing concentration. This is also associated with the highest _Tm as measured by Thermofluor.

Conclusions: During the expression and purification of the humanized Ab969 panel, it was clear that increasing the antibody concentration above 10 mg/ml resulted in rapid precipitation of some humanized antibody grafts. Specifically, Ab969.g1, Ab969.g6 and Ab969.g7 formed precipitates, while Ab969.g2, Ab969.g4 and Ab969.g5 did not form precipitates. This data shows that substitution of glutamine by a lysine residue at position 38 (K38) in the light chain improves antibody stability when concentrated above 10 mg/ml.

In vitro analysis of embodiment 5Ab969.g2

i) The sequence of Ab969.g2

Ab969.g2 contains a gL7 light chain graft as well as a gH2 heavy chain graft. The alignment between the rat antibody (donor) V region sequence and the human germline (recipient) V region sequence and the final humanized sequences for the light chain graft gL7 and the heavy chain graft gH2 are shown in Figure 2A and 2B.

The heavy chain framework residues in graft gH2 are all from human germline genes. The glutamine residue at position 1 of the human heavy chain framework is replaced by glutamic acid (E1) to improve homogeneity, for example by converting glutamine to pyroglutamic acid at the N-terminus of antibodies and antibody fragments. Product expression and purification.

With the exception of residue 71 (Kabat numbering), the light chain framework residues in graft gL7 were all from human germline genes, where the donor residue tyrosine was preserved. Retention of residues provided improved potency of humanized antibodies, as shown in Example 3.

ii) Inhibition of IL-34-dependent activation of human monocytes

The activity of Ab969.g2 compared to 969.g0 was assessed in an IL-34 dependent human monocyte assay. Experiments were performed using two separate monocyte donors and the mean _IC50 for inhibition of IL-34-mediated monocyte stimulation was calculated (Table 12). There was no significant difference in _IC50 between Ab969.g2 and Ab969.g0.

In the presence of 100 ng/ml recombinant human IL-34 and dose titration of anti-CSF-1R antibody (semi-log serial dilution, including 16 concentrations, the maximum concentration is μg/ml), primary human monocytes were cultured at 20,000 cells/well were seeded. The cells were incubated for 24 hours and the supernatant collected. Secreted MCP-1 was measured by ELISA (R&Dsystems DY279). Graphs show percent inhibition of MCP-1 production compared to CSF-1 only controls.

Table 12

iii) Binding of Ab969.g2 to SNPs of human CSF-1R

Four non-synonymous single nucleotide polymorphisms (SNPs) in the ligand binding domain of the human CSF-IR gene have been reported to exist in the human population. These SNPs were V32G, A245S, H247P and V279M (Fig. IF). Individual SNP variants of human CSF-IR were generated and stably expressed in cell lines. Binding of Ab969.g2 to each SNP was confirmed by flow cytometry.

Example 6 Pharmacodynamic marker analysis in cynomolgus monkeys

A pharmacokinetic/pharmacodynamic (PK/PD) study using the humanized monoclonal anti-CSF-1R antibody 969.g2 was performed to demonstrate the pharmacological activity of the antibody in non-human primates (cynomolgus monkeys) . Groups of three cynomolgus monkeys were administered a single dose of 969.g2 antibody intravenously at 7 mg/kg (group 1 ) or 1.5 mg/kg (group 2). Antibodies were well tolerated with no adverse clinical signs. Serum samples were collected at various time points during the 25-day study.

Serum concentrations of 969.g2 were measured by ELISA and pharmacokinetic analysis was performed (Table 13). PK parameters were calculated using WinNonlin software.

t _1/2 is the half-life of the antibody in serum.

C _max is the peak concentration of antibody in serum.

AUC is the area under the curve (integration of the concentration-time curve) and provides an indication of the total exposure to the drug.

Clearance is the volume of plasma that clears a drug per unit time.

Vol.Dist. is the volume of distribution, which is the apparent volume of drug distribution.

There was a good correlation between observed and predicted values (animal 2 was the exception and considered an outlier). _Cmax was dose proportional; AUC was greater than dose proportional, indicating lower clearance at higher doses. Most of 969.g2 was detected in serum.

Table 13

The major route for clearance of CSF-1 is through binding to its cognate receptor, CSF-1R. Blockade of CSF-1 binding to CSF-1R is expected to increase serum concentrations of CSF-1 by preventing receptor-mediated clearance; a phenomenon that has been observed in murine models. Thus, an increase in serum CSF-1 concentration is a pharmacodynamic marker of CSF-1R binding and inhibition.

Both doses of 969.g2 resulted in a rapid and significant accumulation of serum CSF-1. The effect was dose dependent, with the 7 mg/kg dose providing approximately 10-fold higher peak CSF-1 concentrations than the 1.5 mg/kg dose. The observed pharmacokinetic/pharmacodynamic relationship followed antibody clearance by normalization of CSF-1 levels. For both treatment groups, CSF-1 levels returned to baseline at the end of the study. The results are shown in Figures 15a and 15b.

Figure 15a shows CSF-1 concentrations in serum samples collected from cynomolgus monkeys treated with a single iv dose of 7 mg/kg Ab969.g2. Serum concentrations of CSF-1 were measured in two independent assays. The graph shows the mean CSF-1 concentration with standard error (squares) for three animals in Group 1 (7 mg/kg). Mean serum concentrations of Ab969.g2 are also shown (circles).

Figure 15b shows CSF-1 concentrations in serum samples collected from cynomolgus monkeys treated with a single iv dose of 1.5 mg/kg Ab969.g2. Serum concentrations of CSF-1 were measured in two independent assays. The graph shows the mean CSF-1 concentration of three animals in group 2 (1.5 mg/kg) with standard error (squares). Mean serum concentrations of Ab969.g2 are also shown (circles).

There are two major populations of circulating human and cynomolgus monocytes; (i) CD14+CD16- "typical" monocytes and (ii) CD14+CD16+ "atypical" or "resident" monocytes. Murine models have demonstrated that resident tissue macrophages, including TAMs, are derived from atypical monocyte populations. Furthermore, atypical monocytes are derived from the further differentiation of a typical monocyte population. Circulating monocyte populations in cynomolgus monkeys dosed with 969.g2 were monitored by four-color flow cytometry of whole blood. Flow cytometry was performed using anti-cynomolgus-CD45-PerCP, anti-human-HLA-DR-APC, anti-human-CD14-FITC (My4 clone), and anti-human-CD16-PE (3G8 clone) antibodies. Use the appropriate isotype control for each antibody to set the gate. Typical monocytes were defined as CD45 ⁺ HLA-DR ⁺ CD14 ⁺ CD16 ^- . Atypical monocytes were defined as CD45 ⁺ HLA-DR ⁺ CD14 ⁺ CD16 ⁺ .

Both doses of 969.g2 induced progressive depletion of atypical CD14 ⁺ CD16 ⁺ monocytes during the first week of the study. Almost total depletion of atypical monocytes was induced by the 7 mg/kg dose. Atypical monocytopenia by the 7 mg/kg and 1.5 mg/kg doses appeared to peak at the day 4 time point, with numbers returning to normal by day 11. The results are shown in Figure 15c. Bar graphs show mean numbers of circulating atypical CD14 ⁺ CD16 ⁺ monocytes with standard errors at time points throughout the study (Day 0, 1 , 4, 18 and 25 (D)). Mean concentrations and standard errors of serum CSF-1 are also plotted.

This example demonstrates (i) the ability of antibody 969.g2 to bind CSF-1R and block CSF-1 binding in cynomolgus monkeys, (ii) the pharmacological activity of antibody 969.g2 in a non-human primate model , (iii) demonstrated that antibody 969.g2 selectively depleted in vivo an atypical population of cynomolgus monocytes - believed to be precursors of tumor-associated macrophages, and (iv) that serum CSF-1 concentrations were suitable as a target for use as Biomarkers used to measure 969.g2 activity.

Example 7: Inhibition of Growth of MCF-7 Breast Cancer Xenografts

Antibody 969.g2 was unable to bind mouse CSF-1R. Therefore, an in vivo mouse study was performed using anti-mouse CSF-1R antibody Ab535 to show the utility of Ab969.g2 in the treatment of cancer and fibrosis. Ab535 has been shown in a number of in vitro experiments to have comparable properties and activities to Ab969.g2.

Ab535 has been shown to inhibit CSF-1 mediated monocyte survival in Example 2iii), with comparable affinity in Example 2iv) and does not trigger CSF-1R internalization in Example 2vii).

As with the in vivo studies performed on Ab535, the in vivo MCF-7 breast cancer xenograft model can be performed as follows:

This study measured the therapeutic efficacy of antibody Ab535 administered subcutaneously (s.c.) relative to control antibody, positive control and vehicle control in immunodeficient nude mice bearing subcutaneous grafts of the human breast cancer xenograft MCF-7. Tumor growth inhibition was used as a treatment parameter. Human breast cancer MCF-7 was used as a subcutaneous xenograft model in immunodeficient female NMRI:nu/nu mice. The MCF-7 cell line was obtained from the National Cancer Institute (USA) tumor bank. For experimental purposes, cells were cultured in RPMI1640 medium + 10% FCS in vitro. Cells were harvested from subconfluent cultures and inoculated subcutaneously into mice. At palpable tumor size (4 to 10 mm), treatment was initiated. Test compounds and vehicle controls were administered (s.c.) three times per week. Positive controls were administered intravenously once daily on days 24, 33 and 40. Injection volumes were adjusted individually for body weight at the time of injection. Tumor diameters were measured with calipers three times a week. Tumor volumes were calculated according to V=(length x (width)2)/2. For calculation of relative tumor volume (RTV), the volume on each measurement day was correlated to the first treatment day. On each measurement day, the median and mean tumor volumes were calculated for each group as well as the percent median of the treated versus control (T/C) values.

The results are shown in Figure 16. Ab535 showed a dose-dependent antitumor effect. Neither dose of the control mouse IgG antibody caused tumor growth inhibition. Relative tumor volumes were comparable to vehicle controls and positive controls that caused tumor growth inhibition.

Conclusions: The test antibody Ab535 elicited a statistically significant antitumor effect at the highest dose (30 mg/kg/day) in the human breast cancer xenograft MCF-7.

Example 8: Inhibition of the growth of PC-3 prostate cancer in situ

The antitumor and antimetastatic efficacy of antibody Ab535 was also tested in vivo using the orthotopic prostate cancer model PC-3. The PC-3 cell line was genetically altered to continuously express luciferase, allowing in vivo bioluminescence imaging analysis, which allowed in vivo monitoring of tumor growth and ex vivo metastasis analysis in selected organs.

The study consisted of 6 experimental groups each containing 11 (group 5) or 12 (all other groups) male NMRI nude mice after randomization. On day 0, PC-3 cells were orthotopically implanted into the prostate of all participating male NMRI nude mice. On day 3, onset of tumor growth was verified via in vivo bioluminescence imaging. On day 8, a second in vivo bioluminescence imaging was performed and tumor bearing animals were randomly divided into six groups based on the imaging results so that the mean bioluminescence intensity and thus tumor size were similar in each group. On the latter day (day 9), treatment was initiated. Animals in groups 2 and 3 received 30 and 10 mg/kg of control antibody, respectively, subcutaneously three times a week until day 42. Animals in treatment groups 4 and 5 received 30 and 10 mg/kg of antibody Ab535, respectively, subcutaneously 3 times a week until day 42. Animals in Group 1 represented vehicle controls and received vehicle (PBS), administered subcutaneously three times per week until day 42. Animals in Group 6 represented the positive control and received 360 mg/kg control intravenously once a week for four weeks (on days 10, 17, 24 and 31). Orthotopically implanted PCs were monitored in vivo using bioluminescent imaging on days 3, 8 (randomization), 15, 22, 29, 36, and 43 during the study -3 tumor growth.

Necropsy was performed at the end of the study. Initial tumor weight and volume were determined. Selected organs (liver, spleen and lung) were harvested, a portion of each was fixed in formalin and the rest were analyzed for metastatic patterns via bioluminescent imaging using an in vitro luciferase assay. In addition, the same sections of the femur and lumbar spine from one leg were collected for analysis of metastatic patterns in the bone using an in vitro luciferase assay.

The in vivo bioluminescent signal consisted of both primary tumors and metastases (whole body imaging). Based on these data, tumor growth curves could be calculated for all groups (Figure 17). Tumor development was uniform across all study groups until randomization and initiation of treatment. Next, the vehicle control group 1 and the two control antibody RTE11 groups 2 and 3 showed regular tumor growth. In the case of the positive control (Group 6), a highly significant slowing of tumor growth measured in vivo at day 43 could be observed. Antibody Ab535 administered at 30 or 10 mg/kg respectively (Groups 4 and 5) resulted in a significant slowing of tumor growth when monitored using in vivo bioluminescence imaging.

Initial tumor volume and wet weight were determined during necropsy on day 44. The positive control (Group 6) resulted in a highly significant reduction in initial tumor volume and weight. Antibody Ab535 was administered at 30 mg/kg (group 4), and a significant reduction in initial tumor volume and weight was observed. When Ab535 was administered at 10 mg/kg (group 5), the reduction in tumor volume was significant but less than that shown by group 4. For the control antibody, no significant antitumor efficacy was observed.

Using bioluminescence imaging, initial tumor luciferase activity was measured after necropsy. The results obtained were comparable to those of necropsy findings and in vivo growth curves. The positive control (Group 6) resulted in a highly significant reduction in initial tumor luciferase activity. Antibody Ab535 administered at 30 mg/kg (Group 4) resulted in a significant reduction in initial tumor luciferase activity, while the reduction was lower when Ab535 was administered at 10 mg/kg (Group 5). For the control antibody, no significant reduction in luciferase activity was observed.

Several other organs (liver, spleen, lung, femur, and part of the lumbar spine) were analyzed using ex vivo bioluminescence imaging. For the positive controls, the femur and lumbar spine could show a significant reduction in luciferase activity, while for the liver and spleen the signal reduction was just below significance. For antibody Ab535 (Group 4) administered at 30 mg/kg, the reduction of luciferase activity in the liver was significant and was significant for all other organs. Control antibodies (Group 2 and Group 3) did not cause any reduction in luciferase activity in all organs tested.

In conclusion, the significant anti-tumor potency of antibody Ab535 in this tumor model as well as the significant anti-metastatic potency could be demonstrated.

Example 9: Effect of Anti-CSF-1R Antibody in Bleomycin-Induced In Vivo Model of Pulmonary Fibrosis

Bleomycin is an antibiotic first isolated from Streptomyces verticillatus and has been used as a chemotherapeutic agent for various cancers. The bleomycin model of pulmonary fibrosis is a well established model and was used essentially as described in Madtes, DK et al., 1999, AmJ RespirCellMolBiol, 20, 924-34. Detailed protocols can be found in Morschl, E., Molina, J.G., Volmer, J., Mohsenin, A., Pero, R.S., Hong, J.S., Kheradmand, F., Lee, J.J. and Blackburn, M.R. (2008), A3adenosine receptor signaling influences pulse monary inflammation and fibrosis. Am. J. Respir. Cell Mol. Biol. 39:697-705.

All mice used were wild-type C57Blk6 female mice (20 g) purchased from Harlan Labs. Intratracheal (IT) instillation was used in which mice were anesthetized with avertin and a tracheostomy was performed to instill 3.5 unit doses of bleomycin in 50 μl saline or 50 μl saline alone as a control. Treatment in this manner resulted in a peak inflammatory phase at day 7 post-bleomycin exposure and a maximal fibrotic phase at day 21 post-exposure. The effect of antibody Ab535 was studied where the antibody was administered only in the fibrotic phase of the model and administered subcutaneously at 30 mg/kg three times a week from days 9-21. Animals were sacrificed on day 21 and readouts included histopathological analysis of lungs, BAL (bronchoalveolar lavage) fluid cellularity and measurements of soluble collagen in BAL fluid. Histopathological analysis was performed to score lung lesions using the modified Ehrlich scoring system to determine the severity of pulmonary fibrosis (Hubner RH et al., 2008, Biotechniques 44:507-17). Excised lungs were inflated to 25 cm pressure with 10% formalin and processed through a series of alcohols and xylenes, embedded in paraffin, and tissue sections were deparaffinized prior to processing and stained with Masson's trichrome To stain.

Estimate the amount of soluble collagen in the BAL fluid using a commercially available Sircol assay kit following the manufacturer's instructions.

The effect on macrophage populations in BAL fluid was determined using cytospin preparations. Aliquots of BAL cells were thrown onto microscope slides, stained with Diff-Quick and macrophages were counted.

It was found that therapeutic treatment with anti-CSF-1R antibody Ab535, administered starting on day 9, resulted in a dramatic reduction in bleomycin-induced pulmonary fibrosis. Both the severity and extent of fibrosis were significantly reduced. Treated mice had reduced collagen production, improved fibrotic pathology, and improved lung barrier protection.

Figure 18a shows the effect of Ab535 treatment on bleomycin-induced lung fibrosis on BALF collagen concentration compared to isotype control.

Figure 18b shows that treatment of bleomycin-induced lung fibrosis with Ab535 reduces the Ehrlich score of the samples compared to treatment with isotype control, thus showing that mice treated with Ab535 have improved fibrotic pathology.

Figure 18c shows that treatment of bleomycin-induced lung fibrosis with Ab535 reduces albumin concentration in serum compared to treatment with isotype control, which thus shows improved vascular permeability in mice treated with Ab535.

Figure 19 shows representative images from histopathological analysis of lungs from saline control, bleomycin+isotype control and bleomycin+Ab535 treated animals. Animals treated with Ab535 had greatly reduced pulmonary fibrosis compared to isotype control, bleomycin-treated animals.

Example 10 Effect of Anti-CSF-1R Antibody in Adenosine Deaminase Deficient Model of Pulmonary Fibrosis

Adenosine is a potent signaling nucleoside whose levels are elevated when cells are subjected to stress or injury and produces a wide variety of responses when adenosine engages its specific G protein-coupled receptors. Adenosine deaminase (ADA) is a purine metabolizing enzyme that converts adenosine to inosine. ADA knockout mice have been generated and shown to have elevated adenosine levels in serum as well as in tissues such as kidney, liver and lung (Blackburn, MR et al., 1998, J Biol Chem, 273(9):5093-5100). These mice exhibit features of chronic lung disease, such as alveolar destruction, airway inflammation, and excess mucus production, which are associated with elevated levels of adenosine in the lung (Blackburn MR et al., 2000, JExpMed, 192:159-70). These effects resulted in the death of the mice at three weeks of age due to respiratory distress. Exogenous ADA administration using a low-dose regimen reduces adenosine levels and prolongs the lifespan of these mice, allowing the development of a model of pulmonary fibrosis (Chunn JL et al., 2005, J Immunol 175:1937-46, Pedrosa M et al., 2011, PLoS One, 6 (7):e22667). In this model, a slow increase in adenosine levels is associated with elevated profibrotic mediators in the lung including TGFβ-1, increased collagen deposition in lung tissue, and increased pulmonary fibrotic pathology. To investigate the role of molecules with anti-fibrotic potential, ADA-deficient mice were maintained on a low-dose exogenous ADA regimen for several weeks, after which ADA treatment was discontinued and potential anti-fibrotic agents were administered.

The effect of the anti-CSF-1R antibody Ab535 was investigated in an ADA knockout mouse model of pulmonary fibrosis. In this model, enzyme-deficient mice are maintained on ADAase therapy from postnatal days 1-21. ADA-polyethylene glycol (PEG) conjugates (YoungHW et al., 2004, JImmunol 173: 1380-89) were prepared and administered intramuscularly at postnatal days 1, 5, 9, 13 and 17 (0.625, 1.25, 2.5, 2.5 and 2.5 units), followed by an intraperitoneal injection of 5 units on day 21. No further enzymes were administered after day 21. Ab535 was administered subcutaneously at 30 mg/kg in a volume of 100 μl three times a week from day 25 (postnatal) until animals were sacrificed on day 42.

Animals were sacrificed on day 42 and readouts included histopathological analysis of lungs, BAL fluid cellularity and quantification of soluble collagen in BAL fluid. Histopathological analysis was performed to score lung lesions using the modified Ehrlich scoring system to determine the severity of pulmonary fibrosis (Hubner et al., 2008, Biotechniques 44:507-17). Excised lungs were inflated to 25 cm pressure with 10% formalin and processed through a series of alcohols and xylenes, embedded in paraffin, and tissue sections were deparaffinized prior to processing and stained with Masson's trichrome To stain.

The amount of soluble collagen in the BAL fluid was also assessed using a commercially available Sircol assay kit following the manufacturer's instructions.

The effect on macrophage populations in BAL fluid was determined using cytospin preparations. Aliquots of BAL cells were thrown onto microscope slides, stained with Diff-Quick and macrophages were counted.

They found that therapeutic treatment with the anti-CSF-1R antibody Ab535 significantly reduced lung fibrosis in ADA-deficient mice. Treated mice had reduced collagen production, improved fibrotic pathology, and improved lung barrier function and protection.

Figure 20a shows that Ab535 treatment of ADA deficient mice with induced lung fibrosis reduces BALF collagen concentration compared to treatment with isotype control.

Figure 20b shows that treatment of ADA-deficient mice with induced pulmonary fibrosis by Ab535 reduces the Ehrlich score of the samples compared to treatment with isotype controls, which thus shows that mice treated with Ab535 have improved fibrotic disease Science.

Figure 20c shows that Ab535 treatment of ADA-deficient mice with induced pulmonary fibrosis reduces the concentration of albumin in serum compared to treatment with isotype control, which thus shows vascular permeability in Ab535-treated mice Improved.

Figure 2Od shows that treatment of ADA-deficient mice with induced lung fibrosis receiving Ab535 has reduced numbers of macrophages in BAL fluid.

Figure 21 shows representative images from histopathological analysis of lungs from normal mice (ADA+) and ADA-deficient mice with induced pulmonary fibrosis (ADA-) treated with isotype control or Ab535. In ADA- mice, animals treated with Ab535 had greatly reduced lung fibrosis compared to isotype controls.

Thus, treatment with anti-CSF-1R antibodies has been shown to be effective in treating fibrotic disease in two mouse models of pulmonary fibrosis.

Claims (34)

1. an anti-CSF-1R antibody, it comprises heavy chain, and the variable domain of wherein said heavy chain comprises following at least one: have be directed to CDR-H1 with the CDR of the sequence shown in SEQIDNO:4, have and be directed to CDR-H2 and with the CDR of the sequence shown in SEQIDNO:5 and there is the CDR being directed to CDR-H3 with the sequence shown in SEQIDNO:6.

2. anti-CSF-1R antibody according to claim 1, the variable domain of wherein said heavy chain comprise be directed to CDR-H1 with the sequence shown in SEQIDNO:4, be directed to CDR-H2 with the sequence shown in SEQIDNO:5 and be directed to CDR-H3 with the sequence shown in SEQIDNO:6.

3. an anti-CSF-1R antibody, it comprises light chain, and the variable domain of wherein said light chain comprises following at least one: have be directed to CDR-L1 with the CDR of the sequence shown in SEQIDNO:1, have and be directed to CDR-L2 and with the CDR shown in SEQIDNO:2 and there is the CDR being directed to CDR-L3 with the sequence shown in SEQIDNO:3.

4. according to anti-CSF-1R antibody according to claim 1 or claim 2, its other bag light chain, the variable domain of wherein said light chain comprises following at least one: have be directed to CDR-L1 with the CDR of the sequence shown in SEQIDNO:1, have and be directed to CDR-L2 and with the CDR of the sequence shown in SEQIDNO:2 and there is the CDR being directed to CDR-L3 with the sequence shown in SEQIDNO:3.

5. anti-CSF-1R antibody according to claim 4, the variable domain of wherein said light chain comprise be directed to CDR-L1 with the sequence shown in SEQIDNO:1, be directed to CDR-L2 with the sequence shown in SEQIDNO:2 and be directed to CDR-L3 with the sequence shown in SEQIDNO:3.

6. an anti-CSF-1R antibody, the variable domain of wherein said heavy chain comprises three kinds of CDR, and the sequence of CDR-H1 with there is at least 60% identity or similarity with the sequence shown in SEQIDNO:4, the sequence of CDR-H2 with there is at least 60% identity or similarity with the sequence shown in SEQIDNO:5, and the sequence of CDR-H3 with there is at least 60% identity or similarity with the sequence shown in SEQIDNO:6.

7. anti-CSF-1R antibody according to claim 6, it comprises light chain in addition, the variable domain of wherein said light chain comprises three kinds of CDR, and the sequence of CDR-L1 with there is at least 60% identity or similarity with the sequence shown in SEQIDNO:1, the sequence of CDR-L2 with there is at least 60% identity or similarity with the sequence shown in SEQIDNO:2, and the sequence of CDR-L3 with there is at least 60% identity or similarity with the sequence shown in SEQIDNO:3.

8. anti-CSF-1R antibody according to any one of claim 1 to 5, wherein said heavy chain comprises with the sequence shown in SEQIDNO:23.

9. anti-CSF-1R antibody according to any one of claim 1 to 5, wherein said light chain comprises with the sequence shown in SEQIDNO:15.

10. anti-CSF-1R antibody according to any one of claim 1 to 9, wherein said antibody molecule is selected from the complete antibody molecule with total length heavy chain and light chain or its fragment, such as, be selected from and comprise Fab, modified Fab ', Fab ', F (ab ')₂, Fv, VH, VL and scFv fragment group.

11. 1 kinds of anti-CSF-1R antibody, it has and comprises the heavy chain with the sequence shown in SEQIDNO:23 and comprise with the light chain of the sequence shown in SEQIDNO:15.

12. 1 kinds of anti-CSF-1R antibody, the light-chain variable domain that the variable domain of wherein said light chain comprises the antibody with claim 11 has the sequence of at least 80% identity or similarity, and the heavy chain variable domain that the variable domain of wherein said heavy chain comprises the antibody with claim 11 has the sequence of at least 80% identity or similarity.

13. 1 kinds of anti-CSF-1R antibody, it has and comprises the heavy chain with the sequence shown in SEQIDNO:27 and comprising with the light chain of the sequence shown in SEQIDNO:19.

14. 1 kinds of anti-CSF-1R antibody, wherein said heavy chain and light chain have at least 80% identity or similarity with corresponding heavy chain and the light chain of the antibody of claim 13.

The 15. anti-CSF-1R antibody according to any one of claim 1 to 14, it has connected effector molecule or reporter molecules.

The 16. anti-CSF-1R antibody according to any one of claim 1 to 15, it is 10pM for the binding affinity of people CSF-1R or is less than 10pM.

17. 1 kinds of anti-CSF-1R antibody, it such as has the avidity of 100pM or less, and it intersects the combination of antibody blocking and comprising following 6 kinds of CDR: be directed to CDR-L1 with the sequence shown in SEQIDNO:1, be directed to CDR-L2 with the sequence shown in SEQIDNO:2, be directed to CDR-L3 with the sequence shown in SEQIDNO.3, be directed to CDR-H1 with the sequence shown in SEQIDNO:4, be directed to CDR-H2 with the sequence shown in SEQIDNO:5 and be directed to CDR-H3 with the sequence shown in SEQIDNO:6.

18. anti-CSF-1R antibody according to claim 17, the identical table position of antibody that wherein said antibody blocks with it by combining intersects the described combination of blocking-up.

The DNA sequence dna of 19. 1 kinds of separation, the heavy chain of the antibody of its coding according to any one of claim 1 to 18 and/or light chain.

20. 1 kinds of clones or expression vector, it comprises one or more DNA sequence dnas according to claim 19.

21. carriers according to claim 20, wherein said carrier comprises with the sequence shown in SEQIDNO:28 and/or SEQIDNO:20.

22. 1 kinds of host cells, it comprises one or more clone according to claim 21 or expression vectors.

23. 1 kinds of methods for the preparation of the antibody according to any one of claim 1 to 18, described method comprises the host cell cultivating claim 22 and is separated described antibody.

24. 1 kinds of pharmaceutical compositions, it comprises with pharmacy the antibody according to any one of claim 1 to 18 of one or more combinations in acceptable vehicle, thinner or load agent.

25. pharmaceutical compositions according to claim 24, it comprises other activeconstituents in addition.

26. the antibody according to any one of claim 1 to 18 or the composition according to claim 24 or 25, it is used as medicament.

27. the antibody according to any one of claim 1 to 18 or the composition according to claim 24 or 25, it is used for the treatment of cancer.

28. antibody according to any one of claim 1 to 18 or the composition according to claim 24 or 25, it is used for the treatment of fibrotic disease.

29. antibody according to any one of claim 1 to 18 or the composition according to claim 24 or 25 purposes in the medicine for the preparation for the treatment of or preventing cancer.

30. antibody according to any one of claim 1 to 18 or the composition according to claim 24 or 25 purposes in the medicine for the preparation for the treatment of or prevention of fibrotic diseases.

31. 1 kinds are used for the treatment of the method for the people experimenter suffering from cancer or being in risk of cancer, and described method comprises uses the anti-CSF-1R antibody according to any one of claim 1 to 18 of significant quantity or the composition according to claim 24 or 25 to described experimenter.

32. 1 kinds are used for the treatment of the method for the people experimenter suffering from fibrotic disease or be in fibrotic disease risk, and described method comprises uses the anti-CSF-1R antibody according to any one of claim 1 to 18 of significant quantity or the composition according to claim 24 or 25 to described experimenter.

The 33. described antibody used according to claim 27, purposes according to claim 29 or method according to claim 31, wherein said cancer is selected from: mammary cancer, prostate cancer, osteocarcinoma, colorectal carcinoma, leukemia, lymphoma, skin carcinoma, esophagus cancer, cancer of the stomach, stellate cell cancer, carcinoma of endometrium, cervical cancer, bladder cancer, kidney, lung cancer, liver cancer, thyroid carcinoma, head and neck cancer, carcinoma of the pancreas and ovarian cancer.

The 34. described antibody used according to claim 28, purposes according to claim 30 or method according to claim 32, wherein said fibrotic disease is selected from: pulmonary fibrosis such as idiopathic pulmonary fibrosis and cystic fibrosis, renal fibrosis, liver cirrhosis, endomyocardial fibrosis, mediastinum fibrosis, myelofibrosis, retroperitoneal fibrosis, Progressive symmetric erythrokeratodermia masses of fibres, kidney source sexual system fibrosis, Crohn's disease, keloid, myocardial infarction, scleroderma and joint fibrosis.